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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

In their struggle for life, plants can employ sophisticated strategies to defend themselves against potentially harmful pathogens and insects. One mechanism by which plants can increase their level of resistance is by intensifying the responsiveness of their immune system upon recognition of selected signals from their environment. This so-called priming of defence can provide long-lasting resistance, which is based on a faster and/or stronger defence reaction upon pathogen or pest attack. Priming can target various layers of induced defence that are active during different stages of the plant–attacker interaction. Recent discoveries have extended our knowledge about the mechanistic basis of defence priming and suggest that a primed defence state can be inherited epi-genetically from defence-expressing plants. In this review, we provide an overview of the latest insights about defence priming, ranging from early responses controlled by adjustments in hormone-dependent signalling pathways and availability of signal transduction proteins, to longer lasting mechanisms that involve possible regulation chromatin modification or DNA methylation.

Plants frequently live in environments characterized by the presence of simultaneous and different stresses. The intricate and finely tuned molecular mechanisms activated by plants in response to abiotic and biotic environmental factors are not well understood, and less is known about the integrative signals and convergence points activated by plants in response to multiple (a)biotic stresses. Phytohormones play a key role in plant development and response to (a)biotic stresses. Among these, one of the most important signaling molecules is an oxylipin, the plant hormone jasmonic acid. Oxylipins are derived from oxygenation of polyunsaturated fatty acids. Jasmonic acid and its volatile derivative methyl jasmonate have been considered for a long time to be the bioactive forms due to their physiological effects and abundance in the plant. However, more recent studies showed unambiguously that they are only precursors of the active forms represented by some amino acid conjugates. Upon developmental or environmental stimuli, jasmonates are synthesized and accumulate transiently. Upon perception, jasmonate signal transduction process is finely tuned by a complex mechanism comprising specific repressor proteins which in turn control a number of transcription factors regulating the expression of jasmonate responsive genes. We discuss the latest discoveries about the role of jasmonates in plants resistance mechanism against biotic and abiotic stresses. Finally, the deep interplay of different phytohormones in stresses signaling will be also discussed.

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.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea

A deletion in the high affinity nitrate trasporter NRT2.1 in Arabidopsis results in a reduced susceptibility to Pseudomonas syringae by two different mechanisms, the SA priming and an interference in the effector triggered susceptibility. In the present research we further characterized the metabolic and genetic profiles of the mutant nrt2 in the interaction with P. syringae. Despite the priming found in the SA-dependent pathway, the metabolic changes in nrt2 compared with wild-type plants are more remarkable prior infection. This is associated mainly to a pre-existing over representation of signals attributed to aromatic amino acids and phenylpropanoids in the nrt2. Genomic analysis confirms the implication of aromatic aminoacids and phenylpropanoids, but additionally, suggests a new role in ribosomal proteins as the major changes observed in nrt2 upon infection by the bacterium.

An attack of plants by pathogens or treatment with certain resistance-inducing compounds can lead to the establishment of a unique primed state of defense. Primed plants show enhanced defense reactions upon further challenge with biotic or abiotic stress. Here, we report that the primed state in Arabidopsis (Arabidopsis thaliana) is still functional in the next generation without additional treatment. We compared the reactions of Arabidopsis plants that had been either primed with β-amino-butyric acid (BABA) or with an avirulent isolate of the bacteria Pseudomonas syringae pv tomato (PstavrRpt2). The descendants of primed plants showed a faster and higher accumulation of transcripts of defense-related genes in the salicylic acid signaling pathway and enhanced disease resistance upon challenge inoculation with a virulent isolate of P. syringae. In addition, the progeny of primed plants was also more resistant against the oomycete pathogen Hyaloperonospora arabidopsidis. When transgenerationally primed plants were subjected to an additional priming treatment, their descendants displayed an even stronger primed phenotype, suggesting that plants can inherit a sensitization for the priming phenomenon. Interestingly, this primed to be primed phenotype was much reduced in the Arabidopsis β-amino-butyric acid priming mutant ibs1 (induced BABA sterility1). Our results demonstrate that the primed state of plants is transferred to their progeny and confers improved protection from pathogen attack as compared to the descendants of unprimed plants.

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.

In the present study, we evaluated the role of the defense-related gene OCP3 in callose deposition as a response to two necrotrophic fungal pathogens, Botrytis cinerea and Plectosphaerella cucumerina. ocp3 plants exhibited accelerated and intensified callose deposition in response to fungal infection associated with enhanced disease resistance to the two pathogens. A series of double mutant analyses showed potentiation of callose deposition and the heightened disease resistance phenotype in ocp3 plants required the plant hormone abscisic acid (ABA) and the PMR4 gene encoding a callose synthase. This finding was congruent with an observation that ocp3 plants exhibited increased ABA accumulation, and ABA was rapidly synthesized following fungal infection in wild-type plants. Furthermore, we determined that potentiation of callose deposition in ocp3 plants, including enhanced disease resistance, also required jasmonic acid (JA) recognition though a COI1 receptor, however JA was not required for basal callose deposition following fungal infection. In addition, potentiation of callose deposition in ocp3 plants appeared to follow a different mechanism than that proposed for callose β-amino-butyric acid (BABA)-induced resistance and priming, because ocp3 plants responded to BABA-induced priming for callose deposition and induced resistance of a magnitude similar to that observed in wild-type plants. Our results point to a model in which OCP3 represents a specific control point for callose deposition regulated by JA yet ultimately requiring ABA. These results provide new insights into the mechanism of callose deposition regulation in response to pathogen attack; however the complexities of the processes remain poorly understood.

Callose deposition in Arabidopsis has emerged as a popular model system to quantify activity of plant immunity. However, there has been a noticeable rise in contradicting reports about the regulation of pathogen-induced callose. To address this controversy, we have examined the robustness of callose deposition under different growth conditions and in response to two different pathogen-associated molecular patterns, the flagellin epitope Flg22 and the polysaccharide chitosan. Based on a commonly used hydroponic culture system, we found that variations in growth conditions have a major impact on the plant's overall capacity to deposit callose. This environmental variability correlated with levels of hydrogen peroxide (H2O2) production. Depending on the growth conditions, pretreatment with abscissic acid stimulated or repressed callose deposition. Despite a similar effect of growth conditions on Flg22- and chitosan-induced callose, both responses showed differences in timing, tissue responsiveness, and colocalization with H2O2. Furthermore, mutant analysis revealed that Flg22- and chitosan-induced callose differ in the requirement for the NADPH oxidase RBOHD, the glucosinolate regulatory enzymes VTC1 and PEN2, and the callose synthase PMR4. Our study demonstrates that callose is a multifaceted defense response that is controlled by distinct signaling pathways, depending on the environmental conditions and the challenging pathogen-associated molecular pattern.

Water scarcity and corresponding abiotic drought stress is one of the most important factors limiting plant performance and yield. In addition, plant productivity is severely compromised worldwide by infection with microbial pathogens. Two of the most prominent pathways responsible for drought tolerance and disease resistance to fungal pathogens in Arabidopsis are those controlled by the phytohormones abscisic acid (ABA) and the oxylipin methyl jasmonate (MeJA), respectively. Here, we report on the functional characterization of OCP3, a transcriptional regulator from the homeodomain (HD) family. The Arabidopsis loss-of-function ocp3 mutant exhibits both drought resistance and enhanced disease resistance to necrotrophic fungal pathogens. Double-mutant analysis revealed that these two resistance phenotypes have different genetic requirements. Whereas drought tolerance in ocp3 is ABA-dependent but MeJA-independent, the opposite holds true for the enhanced disease resistance characteristics. These observations lead us to propose a regulatory role of OCP3 in the adaptive responses to these two stresses, functioning as a modulator of independent and specific aspects of the ABA- and MeJA-mediated signal transduction pathways.

The antifungal activity of hexanoic acid on the phytopathogen Botrytis cinerea was studied. This chemical inhibited both spore germination and mycelial growth in vitro in a concentration- and pH-dependent manner, and stopped spore germination at a very early stage, preventing germ-tube development. The minimum fungicidal concentration (MFC) for in vitro spore germination was 16 m m . Hexanoic acid also inhibited in vitro mycelial growth of germinated spores at an MFC of 12 m m . Studies performed to characterize the mechanisms underlying the antimicrobial effect of hexanoic acid showed that it alters fungal membrane permeability. In addition, hexanoic acid treatment increased the levels of spermine, spermidine, putrescine and cadaverine in B. cinerea mycelia. Spray application of hexanoic acid at fungicidal concentrations on 4-week-old tomato plants prior to fungal inoculation reduced necrosis diameter by approximately 60%. Application of the same hexanoic acid concentrations on previously infected plants reduced further necrosis expansion by around 30%. The results suggest that this chemical acts as a preventive and curative fungicide. Interestingly, treatments with hexanoic acid at concentrations below the MFC in hydroponic solution prior to fungal inoculation significantly reduced necrosis area. These results suggest an inducer effect of plant responses for hexanoic acid treatments at these concentrations. Hexanoic acid is a good candidate for safe antifungal treatments for the control of B. cinerea , which is responsible for many economic losses on fruits, vegetables and flowers.

The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighboring the infiltrated areas after 2–3 days post-inoculation) included: (i) inhibition of photosynthesis in terms of photosystem II efficiency; (ii) activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and (iii) accumulation of secondary metabolites in cell walls of the epidermis (lignins) and the apoplast of the mesophyll (phytoalexins). Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid, jasmonic acid, and salicylic acid. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii., This work was supported by FEDER Funds, RECUPERA 2020 [grant number 20134R060 to MB]; Junta de Andalucía [grant number P12-AGR370 to MB]; and Ministerio de Economía y Competitividad [grant number AGL2012-32516 to EL-S]. EG and MP-B were recipient of a JAE-Doc contract funded by CSIC.

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., This work was financially supported by the Plan de Promoción de la Investigación-Universitat Jaume I P1-1B2013-68 and the Spanish Ministry MINECO AGL2012-39923 and AGL2015-64990-2R.

The beneficial effects of direct predation by zoophytophagous biological control agents (BCAs), such as the mirid bug Nesidiocoris tenuis , are well-known. However, the benefits of zoophytophagous BCAs’ relation with host plants, via induction of plant defensive responses, have not been investigated until recently. To date, only the females of certain zoophytophagous BCAs have been demonstrated to induce defensive plant responses in tomato plants. The aim of this work was to determine whether nymphs, adult females, and adult males of N. tenuis are able to induce defense responses in tomato plants. Compared to undamaged tomato plants (i.e., not exposed to the mirid), plants on which young or mature nymphs, or adult males or females of N. tenuis fed and developed were less attractive to the whitefly Bemisia tabaci , but were more attractive to the parasitoid Encarsia formosa . Female-exposed plants were more repellent to B. tabaci and more attractive to E. formosa than were male-exposed plants. When comparing young- and mature-nymph-exposed plants, the same level of repellence was obtained for B. tabaci , but mature-nymph-exposed plants were more attractive to E. formosa . The repellent effect is attributed to the signaling pathway of abscisic acid, which is upregulated in N. tenuis -exposed plants, whereas the parasitoid attraction was attributed to the activation of the jasmonic acid signaling pathway. Our results demonstrate that all motile stages of N. tenuis can trigger defensive responses in tomato plants, although these responses may be slightly different depending on the stage considered. The research leading to these results was partially funded by the Spanish Ministry of Economy and Competitiveness (AGL2011-30538-C03 and AGL2014-55616-C3), the Conselleria d’Agricultura, Pesca i Alimentació de la Generalitat Valenciana, and the Italian Ministry of Education, University and Research (PRIN project 2010CXXHJE_004). The authors thank Javier Calvo (KOPPERT BS) for the supply of insects and Servei Central d’Instrumentació Científica of the Universitat Jaume I for technical support. MN received a PhD grant from the University of Catania (Italy). MP-H received a postdoctoral fellowship (Juan de la Cierva program) from the Spanish Ministry of Economy and Competitiveness.

We have examined the role of the callose synthase PMR4 in basal resistance and beta-aminobutyric acid-induced resistance (BABA-IR) of Arabidopsis thaliana against the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Alternaria brassicicola. Compared to wild-type plants, the pmr4-1 mutant displayed enhanced basal resistance against P. syringae, which correlated with constitutive expression of the PR-1 gene. Treating the pmr4-1 mutant with BABA boosted the already elevated levels of PR-1 gene expression, and further increased the level of resistance. Hence, BABA-IR against P. syringae does not require PMR4-derived callose. Conversely, pmr4-1 plants showed enhanced susceptibility to A. brassicicola, and failed to show BABA-IR. Wild-type plants showing BABA-IR against A. brassicicola produced increased levels of JA. The pmr4-1 mutant produced less JA upon A. brassicicola infection than the wild-type. Blocking SA accumulation in pmr4-1 restored basal resistance, but not BABA-IR against A. brassicicola. This suggests that the mutant's enhanced susceptibility to A. brassicicola is caused by SA-mediated suppression of JA, whereas the lack of BABA-IR is caused by its inability to produce callose. A. brassicicola infection suppressed ABA accumulation. Pre-treatment with BABA antagonized this ABA accumulation, and concurrently potentiated expression of the ABA-responsive ABI1 gene. Hence, BABA prevents pathogen-induced suppression of ABA accumulation, and sensitizes the tissue to ABA, causing augmented deposition of PMR4-derived callose.

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO(3) (-). The V(max) for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO(3) (-) uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO(3) (-) and down-regulated by the N status and by NO(3) (-) itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO(3) (-) net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.

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

Plant responses induced by zoophytophagous plant bugs (Miridae) have not been thoroughly studied. Here, we show that three different zoophytophagous predators (Nesidiocoris tenuis, Macrolophus pygmaeus and Dicyphus maroccanus) have different capacities for the induction of responses in tomato plants, resulting in varying degrees of attractiveness of the plants to pests and natural enemies. Tomato plants punctured by N. tenuis were less attractive to the whitefly Bemisia tabaci and to the lepidopteran Tuta absoluta. In contrast, tomato plants punctured by M. pygmaeus and D. maroccanus were not able to repel B. tabaci and, more interestingly, became more attractive to T. absoluta. The ability of N. tenuis to make tomato plants less attractive to B. tabaci was attributed to the activation of the abscisic acid (ABA) signaling pathway, which was only up-regulated in plants punctured by N. tenuis. However, the phytohormones involved in the behavioral responses of T. absoluta could not be identified; therefore, further studies are required. Additionally, all three zoophytophagous mirid predators activated jasmonic acid (JA) signaling pathways, which resulted in the parasitoid Encarsia formosa being attracted to tomato plants. Here, the implications of these results on the efficacy of these three predators as biocontrol agents are discussed. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under the Grant agreement n°265865 and by the Spanish Ministry of Science and Innovation (AGL2011-30538-C03) and the Conselleria d’Agricultura, Pesca i Alimentació de la Generalitat Valenciana. The authors thank Javier Calvo (KOPPERT BS) for supplying the insects and Universitat Jaume I-SCIC for providing technical support.

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid β-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.

Three novel amides of adipic acid, 5-carbamoil ethyl pentanoate (N1), 5-(2-furfurylmethylcarbamoil) ethyl pentanoate (N2) and 5-(3-aminopropylcarbamoil) ethyl pentanoate (N3) were synthesized and their resistance-inducing activity was studied on pepper (Capsicum annuum) plants. Treatment with low doses of each amide protected pepper plants against the pathogen Alternaria solani, N1 being the most effective. Although their mode of action is still unknown, prechallenge studies demonstrated the induction of the phenylpropanoid pathway and an antisenescence effect. These effects were previously shown for other mixtures and derivatives of adipic acid. These new chemicals showed no antimicrobial activity at doses much higher than those used for plant treatments. This fact supports the idea that they are acting mainly via planta by inducing natural resistance pathways. Treatments based on these novel chemicals produce no phytotoxic effects and improve plant growth and development. For that reason, they could constitute an attractive alternative to fungicides. It is interesting to note that they are adipic acid derivatives, a compound used as alimentary additive with non toxic properties. Obviously a complete safety study is necessary before these chemicals could be commerzialized.

In several plant species, oxidative stress has been shown to be one of the causes of damage produced by salinity. In order to assess the implication of oxidative stress in the reported sensitivity of the citrus rootstock Carrizo citrange to salt stress, 5-month-old seedlings were grown with increasing NaCl concentrations added to the watering solution. As an indicator of oxidative damage, malondialdehyde content was measured. The antioxidant capability of the plants was determined by measuring superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase activities together with the non-enzymatic antioxidant activity. As additional physiological responses to the stress, 1-aminocyclopropane-1-carboxilic acid and proline accumulation were assessed. Data indicate that Carrizo citrange responded to salt-induced oxidative stress by increasing enzymatic and non-enzymatic antioxidant defenses proportionally to the extent of the stress imposed, and that in all plants the malondialdehyde content remained at a moderate level. We suggest that the important deleterious effects reported in Carrizo citrange grown under high NaCl concentrations are mainly due to a cellular intoxication by Cl(-) ions and not to the salt-induced oxidative stress.

1,2,3,4-tetra-O-acetyl-6-ethyladipate-beta- D-glucopyranose (TOGE), a glycoside derivative of adipic acid monoethyl ester and 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose, was synthesized and the resistance-inducing activity shown by TOGE-1 (TOGE plus furfurylamine) and TOGE-2 (TOGE plus 1,3-diaminepropane) was assayed. TOGE-1 and TOGE-2 protected tomato plants against two different fungal pathogens Phytophthora citrophthora and Alternaria solani. Foliar treatments at very low concentrations (2.5 mg l(-1) TOGE, 0.5 mg l(-1) amine) clearly reduced the disease incidence for both pathogens. TOGE-2 application was the most effective on intact plants as well as on detached leaves, reducing fungal growth by more than 46% with respect to control plants. On the other hand TOGE-1 treatment reduced fungal advance by 21%. These results demonstrate a high protective effect against fungal infections for both chemicals. A possible direct antimicrobial effect was discounted due to the weak activity observed in vitro against these pathogens at the low concentrations used in plants. TOGE-2 treatment clearly activates resistance against both pathogens and improves the protective effect previously shown by FGA mixture (adipic acid monoethyl ester, 1,2,3,4-tetra-O-acetyl-beta- D-glucopyranose and furfurylamine) [V. Flors et al. (2001) J Agric Food Chem 49:2569-2575]. The obtained results indicate that TOGE-1 and TOGE-2 act as resistance inducers. Although their mode of action is still unknown, pre-challenge studies have demonstrated the induction of the phenylpropanoid pathway and antioxidant activities. Both chemicals have demonstrated a beneficial effect on plant development, increasing chlorophyll and protein contents, photosynthetic rate and water-use efficiency. The improvement of plant growth and development produced by these treatments suggests crop tolerance to these chemicals, although effective formulations that are safe to humans must be developed before this technology can be used commercially.

Callose is an amorphous homopolymer, composed of β-1, 3-glucan, which is widespread in higher plants. Callose is involved in multiple aspects of plant growth and development. It is synthetized in plants at the cell plate during cytokinesis, in several stages during pollen development and is deposited at plasmodesmata to regulate the cell-to-cell movement of molecules. Moreover, it is produced in response to multiple biotic and abiotic stresses (Chen and Kim, 2009). Callose is considered to act as a physical barrier by strengthening the plant cell well to slow pathogen infection and to contribute to the plant’s innate immunity. Thus the callose staining method is useful to quantify activity of plant immunity. In addition, this staining can be used to visualize structures in plant tissue, where the callose may be implied whether during the development of plants or response against pathogen infection. This method is based on the use of methyl blue which reacts with (13)--glucans to give a brilliant yellow fluorescence in UV light. Moreover, calcofluor stains chitin present in fungal cell membranes and also binds to cellulose at locations where the cuticle is damaged. Authors thank Universitat Jaume I and the National R&D Plan (AGL2010-22300-C03-02, Spain for funding support.

In the last decade, biological control programs for greenhouse tomatoes and other crops have been successfully implemented using zoophytophagous plant bugs (Miridae), which can feed on both plant tissues and insect prey. It is well known that plants respond to herbivore attacks by releasing volatile compounds through diverse pathways triggered by phytohormones. These herbivore-induced plant volatiles can alert neighboring plants, repel or attract herbivores, and attract natural enemies of these herbivores. Nevertheless, the possible benefits of induced plant responses by zoophytophagous predators that could add to their usefulness as biocontrol agents have not been studied until now. Here we show that the zoophytophagous predator Nesidiocoris tenuis activated abscisic acid and jasmonic acid (JA) signaling pathways in tomato plants, which made them less attractive to the whitefly Bemisia tabaci, a major tomato pest worldwide, and more attractive to the whitefly parasitoid, Encarsia formosa. We also found that intact tomato plants exposed to volatiles from N. tenuis-punctured plants activated the JA pathway, and as a consequence, E. formosa was also attracted to these intact plants with activated defense systems. Thus, our results demonstrate that N. tenuis not only benefits tomato plants directly by entomophagy but also indirectly by phytophagy, which induces a physiological response in the tomato plant. The research leading to these results was funded by the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 265865, the Spanish Ministry of Science and Innovation (AGL2011-30538-C03) and the Conselleria d’Agricultura, Pesca i Alimentació de la Generalitat Valenciana. The authors thank Virginia Pedroche (IVIA) and Blas Agut (UJI) for their technical assistance, Javier Calvo (KOPPERT BS) for the supply of insects, Universitat Jaume I-SCIC for technical support and two anonymous reviewers for their valuable comments on a previous version of this manuscript.

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.

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.

The use of wastewater as an alternative water resource for irrigation needs specific studies to evaluate its effect on different soils and crops. Young Citrus trees have been irrigated with wastewater from a sewage treatment plant during three consecutive seasons and the growth, leaf mineral status and fruit quality measured. Some differences have been found between groundwater and wastewater composition. Sodium ion, chloride ion, organic matter and boron concentrations were always higher in wastewater. In contrast, high nitrate levels were found in groundwater, probably due to aquifer contamination. In all seasons the values of soil-analysed elements were within the optimal ranges for this kind of crop and soil. Levels of sodium, chloride and boron in leaf tissue were below the toxic levels for Citrus. Leaf nitrogen content was in the optimal nutrition range and no significant difference was observed between samples from trees irrigated with wastewater and groundwater. Both growth and fruit quality parameters were unaffected by the high levels of sodium, chloride and boron in wastewater. Collectively our data show that no detrimental effects are found after using wastewater for irrigation of young Citrus trees. More importantly, fertiliser rates could be lowered significantly without reducing yields or affecting leaf nutrient levels when using reclaimed wastewater. Thus reclaimed wastewater appears to be a suitable alternative water resource for irrigation of Citrus trees. © 2000 Society of Chemical Industry

Plants activate local and systemic defence mechanisms upon exposure to stress. This innate immune response is partially regulated by plant hormones, and involves the accumulation of defensive metabolites. Although local defence reactions to herbivores are well studied, less is known about the impact of root herbivory on shoot defence. Here, we examined the effects of belowground infestation by the western corn rootworm Diabrotica virgifera virgifera on aboveground resistance in maize. Belowground herbivory by D. v. virgifera induced aboveground resistance against the generalist herbivore Spodoptera littoralis, and the necrotrophic pathogen Setosphaeria turcica. Furthermore, D. v. virgifera increased shoot levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and primed the induction of chlorogenic acid upon subsequent infestation by S. littoralis. To gain insight into the signalling network behind this below- and aboveground defence interaction, we compiled a set of 32 defence-related genes, which can be used as transcriptional marker systems to detect activities of different hormone-response pathways. Belowground attack by D. v. virgifera triggered an ABA-inducible transcription pattern in the shoot. The quantification of defence hormones showed a local increase in the production of oxylipins after root and shoot infestation by D. v. virgifera and S. littoralis, respectively. On the other hand, ABA accumulated locally and systemically upon belowground attack by D. v. virgifera. Furthermore, D. v. virgifera reduced the aboveground water content, whereas the removal of similar quantities of root biomass had no effect. Our study shows that root herbivory by D. v. virgifera specifically alters the aboveground defence status of a maize, and suggests that ABA plays a role in the signalling network mediating this interaction.

Grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The b-glucan laminarin (Lam) and its sulfated derivative (PS3) have been previously demonstrated to induce resistance in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR)-proteins and phytoalexin production, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i) the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii) grapevine immune responses primed by PS3 during P. viticola infection. Our results showed that i) PS3 was unable to elicit reactive oxygen species (ROS) production, cytosolic Ca2+ concentration variations, mitogen-activated protein kinase (MAPK) activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii) PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA) and jasmonate-(JA)-dependent ones. After P. viticola inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Modulation of the hormonal and transcriptional profiles, including changes related to defense signalling, has been reported in many host plants during AM symbioses. These changes have been often related to the improved stress tolerance common in mycorrhizal plants. However, results on the alterations in phytohormones content and their role on the symbiosis are controversial. Here, an integrative analysis of the response of phylogenetically diverse plants (i.e., tomato, soybean, and maize) to two mycorrhizal fungi -Funneliformis mosseae and Rhizophagus irregularis- was performed. The analysis of the defense-related hormones salicylic acid, abscisic acid, and jasmonates, and the expression of marker genes of the pathways they regulate, revealed significant changes in the roots of mycorrhizal plants. These changes depended on both the plant and the AM fungus (AMF) involved. However, general trends can be identified: roots associated with the most effective colonizer R. irregularis showed fewer changes in these defense-related traits, while the colonization by F. mosseae led to significant modifications in all plants tested. The up-regulation of the jasmonate pathway by F. mosseae was found to be highly conserved among the different plant species, suggesting an important role of jasmonates during this AM interaction. Our study evidences a strong influence of the AMF genotype on the modulation of host defense signalling, and offers hints on the role of these changes in the symbiosis.

Selected stimuli can prime the plant immune system for a faster and stronger defense reaction to pathogen attack. Pretreatment of Arabidopsis with the chemical agent β-aminobutyric acid (BABA) augmented H2O2 and callose production after induction with the pathogen-associated molecular pattern (PAMP) chitosan, or inoculation with the necrotrophic fungus Plectosphaerella cucumerina. However, BABA failed to prime H2O2 and callose production after challenge with the bacterial PAMP Flg22. Analysis of Arabidopsis mutants in reactive oxygen species (ROS) production (rbohD) or ROS scavenging (pad2, vtc1, and cat2) suggested a regulatory role for ROS homeostasis in priming of chitosan- and P. cucumerina-inducible callose and ROS. Moreover, rbohD and pad2 were both impaired in BABA-induced resistance against P. cucumerina. Gene expression analysis revealed direct induction of NADPH/respiratory burst oxidase protein D (RBOHD), γ-glutamylcysteine synthetase 1 (GSH1), and vitamin C defective 1 (VTC1) genes after BABA treatment. Conversely, ascorbate peroxidase 1 (APX1) transcription was repressed by BABA after challenge with chitosan or P. cucumerina, probably to provide a more oxidized environment in the cell and facilitate augmented ROS accumulation. Measuring ratios between reduced and oxidized glutathione confirmed that augmented defense expression in primed plants is associated with a more oxidized cellular status. Together, our data indicate that an altered ROS equilibrium is required for augmented defense expression in primed plants.

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.

Prone to attacks by pathogens and pests, plants employ intricate chemical defense mechanisms consisting of metabolic adaptations. However, many plant attackers are manipulating the host metabolism to counteract defense responses and to induce favorable nutritional conditions. Advances in analytical chemistry have allowed the generation of extensive metabolic profiles during plant-pathogen and pest interactions. Thereby, metabolic processes were found to be highly specific for given tissues, species, and plantpathogen/pest interactions. The clusters of identified compounds not only serve as base in the quest of novel defense compounds, but also as markers for the characterization of the plants’ defensive state. The latter is especially useful in agronomic applications where meaningful markers are essential for crop protection. Cereals such as maize make use of their metabolic arsenal during both local and systemic defense responses, and the chemical response is highly adapted to specific attackers. Here, we summarize highlights and recent findings of metabolic patterns of cereals under pathogen and pest attack. National Centre of Competence in Research (NCCR) 'Plant Survival' SNF 31003A_140593 Swiss National Science Foundation Plan de Promocion de la Investigacion de la Universitat Jaume I P1.1B2010-06

Systemic acquired resistance (SAR) is a plant immune response to pathogen attack. Recent evidence suggests that plant immunity involves regulation by chromatin remodeling and DNA methylation. We investigated whether SAR can be inherited epigenetically following disease pressure by Pseudomonas syringae pv tomato DC3000 (PstDC3000). Compared to progeny from control-treated Arabidopsis (Arabidopsis thaliana; C1), progeny from PstDC3000-inoculated Arabidopsis (P1) were primed to activate salicylic acid (SA)-inducible defense genes and were more resistant to the (hemi)biotrophic pathogens Hyaloperonospora arabidopsidis and PstDC3000. This transgenerational SAR was sustained over one stress-free generation, indicating an epigenetic basis of the phenomenon. Furthermore, P1 progeny displayed reduced responsiveness of jasmonic acid (JA)-inducible genes and enhanced susceptibility to the necrotrophic fungus Alternaria brassicicola. This shift in SA- and JA-dependent gene responsiveness was not associated with changes in corresponding hormone levels. Instead, chromatin immunoprecipitation analyses revealed that SA-inducible promoters of PATHOGENESIS-RELATED GENE1, WRKY6, and WRKY53 in P1 plants are enriched with acetylated histone H3 at lysine 9, a chromatin mark associated with a permissive state of transcription. Conversely, the JA-inducible promoter of PLANT DEFENSIN1.2 showed increased H3 triple methylation at lysine 27, a mark related to repressed gene transcription. P1 progeny from the defense regulatory mutant non expressor of PR1 (npr1)-1 failed to develop transgenerational defense phenotypes, demonstrating a critical role for NPR1 in expression of transgenerational SAR. Furthermore, the drm1drm2cmt3 mutant that is affected in non-CpG DNA methylation mimicked the transgenerational SAR phenotype. Since PstDC3000 induces DNA hypomethylation in Arabidopsis, our results suggest that transgenerational SAR is transmitted by hypomethylated genes that direct priming of SA-dependent defenses in the following generations.

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

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.

RNA–directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plantspecific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)–mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)–mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA–related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear., We acknowledge the financial support of the Spanish MICINN (grants BFU2009-09771 and Consolider-TRANSPLANTA to PV) and Generalitat Valenciana (Prometeo2010/020 to PV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In the present study, we evaluated the role of the defense-related gene OCP3 in callose deposition as a response to two necrotrophic fungal pathogens, Botrytis cinerea and Plectosphaerella cucumerina. ocp3 plants exhibited accelerated and intensified callose deposition in response to fungal infection associated with enhanced disease resistance to the two pathogens. A series of double mutant analyses showed potentiation of callose deposition and the heightened disease resistance phenotype in ocp3 plants required the plant hormone abscisic acid (ABA) and the PMR4 gene encoding a callose synthase. This finding was congruent with an observation that ocp3 plants exhibited increased ABA accumulation, and ABA was rapidly synthesized following fungal infection in wild-type plants. Furthermore, we determined that potentiation of callose deposition in ocp3 plants, including enhanced disease resistance, also required jasmonic acid (JA) recognition though a COI1 receptor, however JA was not required for basal callose deposition following fungal infection. In addition, potentiation of callose deposition in ocp3 plants appeared to follow a different mechanism than that proposed for callose β-amino-butyric acid (BABA)-induced resistance and priming, because ocp3 plants responded to BABA-induced priming for callose deposition and induced resistance of a magnitude similar to that observed in wild-type plants. Our results point to a model in which OCP3 represents a specific control point for callose deposition regulated by JA yet ultimately requiring ABA. These results provide new insights into the mechanism of callose deposition regulation in response to pathogen attack; however the complexities of the processes remain poorly understood.

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses. Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1 , were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2 , PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea , it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment. We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.

Benzoxazinoids (BXs), such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. The first step in BX biosynthesis converts indole-3-glycerol phosphate into indole. In maize (Zea mays), this reaction is catalyzed by either BENZOXAZINELESS1 (BX1) or INDOLE GLYCEROL PHOSPHATE LYASE (IGL). The Bx1 gene is under developmental control and is mainly responsible for BX production, whereas the Igl gene is inducible by stress signals, such as wounding, herbivory, or jasmonates. To determine the role of BXs in defense against aphids and fungi, we compared basal resistance between Bx1 wild-type and bx1 mutant lines in the igl mutant background, thereby preventing BX production from IGL. Compared to Bx1 wild-type plants, BX-deficient bx1 mutant plants allowed better development of the cereal aphid Rhopalosiphum padi, and were affected in penetration resistance against the fungus Setosphaeria turtica. At stages preceding major tissue disruption, R. padi and S. turtica elicited increased accumulation of DIMBOA-glucoside, DIMBOA, and 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucoside (HDMBOA-glc), which was most pronounced in apoplastic leaf extracts. Treatment with the defense elicitor chitosan similarly enhanced apoplastic accumulation of DIMBOA and HDMBOA-glc, but repressed transcription of genes controlling BX biosynthesis downstream of BX1. This repression was also obtained after treatment with the BX precursor indole and DIMBOA, but not with HDMBOA-glc. Furthermore, BX-deficient bx1 mutant lines deposited less chitosan-induced callose than Bx1 wild-type lines, whereas apoplast infiltration with DIMBOA, but not HDMBOA-glc, mimicked chitosan-induced callose. Hence, DIMBOA functions as a defense regulatory signal in maize innate immunity, which acts in addition to its well-characterized activity as a biocidal defense metabolite.

Arbuscular mycorrhizal (AM) fungi colonize the roots of most plants to establish a mutualistic symbiosis leading to important benefits for plant health. We have recently shown that AM symbiosis alters both transcriptional and hormonal profiles in tomato roots, many of these changes related to plant defense. Here, we analytically demonstrate that the levels of other important defense-related compounds as phenolic acids are also altered in the symbiosis. Both caffeic and chlorogenic acid levels significantly decreased in tomato roots upon mycorrhization, while ferulic acid increased. Moreover, in the case of caffeic acid a differential reduction was observed depending on the colonizing AM fungus. The results confirm that AM associations imply the regulation of plant defense responses, and that the host changes may vary depending on the AM fungus involved. The potential implications of altered phenolic acid levels on plant control over mycorrhizal colonization and in the plant resistance to pathogens is discussed.

Supported by grants AGL2006-08029 from the National R&D Plan of the MINCIN and PERG- 02-2007-224751 from the Marie Curie programme from the European Commission, Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. The symbiosis significantly affects the host physiology in terms of nutrition and stress resistance. Despite the lack of host range specificity of the interaction, functional diversity between AM fungal species exists. The interaction is finely regulated according to plant and fungal characters, and plant hormones are believed to orchestrate the modifications in the host plant. Using tomato as a model, an integrative analysis of the host response to different mycorrhizal fungi was performed combining multiple hormone determination and transcriptional profiling. Analysis of ethylene-, abscisic acid-, salicylic acid-, and jasmonate-related compounds evidenced common and divergent responses of tomato roots to Glomus mosseae and Glomus intraradices, two fungi differing in their colonization abilities and impact on the host. Both hormonal and transcriptional analyses revealed, among others, regulation of the oxylipin pathway during the AM symbiosis and point to a key regulatory role for jasmonates. In addition, the results suggest that specific responses to particular fungi underlie the differential impact of individual AM fungi on plant physiology, and particularly on its ability to cope with biotic stresses

We have demonstrated that root treatment with hexanoic acid protects tomato plants against Botrytis cinerea. Hexanoic acid-induced resistance (Hx-IR) was blocked in the jasmonic acid (JA)-insensitive mutant jai1 (a coi1 homolog) and in the abscisic acid (ABA)-deficient mutant flacca (flc). Upon infection, the LoxD gene as well as the oxylipin 12-oxo-phytodienoic acid and the bioactive molecule JA-Ile were clearly induced in treated plants. However, the basal ABA levels were not altered. Hexanoic acid primed callose deposition against B. cinerea in a cultivar-dependent manner. Treated plants from Ailsa Craig, Moneymaker, and Rheinlands Ruhm showed increased callose deposition but not from Castlemart. Hexanoic acid did not prime callose accumulation in flc plants upon B. cinerea infection; therefore, ABA could act as a positive regulator of Hx-IR by enhancing callose deposition. Furthermore, although hexanoic acid protected the JA-deficient mutant defensless1 (def1), the priming for callose was higher than in the wild type. This suggests a link between JA and callose deposition in tomato. Hence, the obtained results support the idea that callose, oxylipins, and the JA-signaling pathway are involved in Hx-IR against B. cinerea. Moreover our data support the relevance of JA-signaling for basal defense against this necrotroph in tomato. Hexanoic acid also protected against Pseudomonas syringae, indicating a broad-spectrum effect for this new inducer.

Long known only for its role in abiotic stress tolerance, recent evidence shows that abscisic acid (ABA) also has a prominent role in biotic stress. Although it acts as a negative regulator of disease resistance, ABA can also promote plant defense and is involved in a complicated network of synergistic and antagonistic interactions. Its role in disease resistance depends on the type of pathogen, its specific way of entering the host and, hence, the timing of the defense response and the type of affected plant tissue. Here, we discuss the controversial evidence pointing to either a repression or a promotion of resistance by ABA. Furthermore, we propose a model in which both possibilities are integrated. © 2009 Elsevier Ltd. All rights reserved.