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[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress., Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.

New strategies of control need to be developed with the aim of economic and environmental sustainability in plant and crop protection. Metabolomics is an excellent platform for both understanding the complex plant–pathogen interactions and unraveling new chemical control strategies. GC-MS-based metabolomics, along with a phytohormone analysis of a compatible and incompatible interaction between tomato plants and Fusarium oxysporum f. sp. lycopersici, revealed the specific volatile chemical composition and the plant signals associated with them. The susceptible tomato plants were characterized by the over-emission of methyl- and ethyl-salicylate as well as some fatty acid derivatives, along with an activation of salicylic acid and abscisic acid signaling. In contrast, terpenoids, benzenoids, and 2-ethylhexanoic acid were differentially emitted by plants undergoing an incompatible interaction, together with the activation of the jasmonic acid (JA) pathway. In accordance with this response, a higher expression of several genes participating in the biosynthesis of these volatiles, such as MTS1, TomloxC,TomloxD, and AOS, as well as JAZ7, a JA marker gene, was found to be induced by the fungus in these resistant plants. The characterized metabolome of the immune tomato plants could lead to the development of new resistance inducers against Fusarium wilt treatment.

This article belongs to the Section Molecular Plant Sciences., Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions., This work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) Grants BIO2017-88321-R (V.P.) and PID2019-104126RB-I00 (G.G.).

© 2020 by the authors., Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

[EN] Background: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. Results: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1- silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. Conclusions: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection., This work was supported by grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, Spanish Ministry of Science and Innovation, and grant AICO/2017/048 from the Valencian Local Government (Generalitat Valenciana, Spain). LC was supported by a predoctoral fellowship (ACIF/2010/231) from the Valencian Local Government (Generalitat Valenciana, Spain). None of the funding bodies was involved in the design of the study, the collection, analysis, and interpretation of the data, nor in the writing of the manuscript, which was made entirely by the authors.

© The Author(s)., Benzothiadiazole (BTH) is a functional analogue of the phytohormone salycilic acid (SA) involved in the plant immune response. NahG tomato plants are unable to accumulate SA, which makes them hypersusceptible to several pathogens. Treatments with BTH increase the resistance to bacterial, fungal, viroid, or viral infections. In this study, metabolic alterations in BTH-treated Money Maker and NahG tomato plants infected by citrus exocortis viroid (CEVd) were investigated by nuclear magnetic resonance spectroscopy. Using multivariate data analysis, we have identified defence metabolites induced after viroid infection and BTH-treatment. Glycosylated phenolic compounds include gentisic and ferulic acid accumulated in CEVd-infected tomato plants, as well as phenylalanine, tyrosine, aspartate, glutamate, and asparagine. Besides, an increase of γ-aminobutyric acid (GABA), glutamine, adenosine, and trigonelline, contributed to a clear discrimination between the metabolome of BTH-treated tomato leaves and their corresponding controls. Among them, GABA was the only metabolite significantly accumulated in both genotypes after the chemical treatment. In view of these results, the addition of GABA was performed on tomato plants infected by CEVd, and a reversion of the NahG hypersusceptibility to CEVd was observed, indicating that GABA could regulate the resistance to CEVd induced by BTH., This research was funded by Dirección General de Programas y Transferencia de Conocimiento, from the Spanish Ministry of Science and Innovation, Grant BIO2012-33419. MP. López-Gresa was the recipient of a postdoctoral fellowship JC2008-00432 Spanish Ministry of Science and Innovation.

© 2020 by the authors., Agronomy solutions for modifying pre-harvest grape ripening are needed for a more sustainable viticulture. Field experiments were performed in Vitis vinifera L. vines to study the effect of the previously described stomata-closing compound (Z)-3-hexenyl butyrate (HB). Exogenous treatments at different doses were periodically carried out using a randomized block design. Firstly, we observed that HB was able to induce stomatal closure in grapevine plants. Under field conditions, the application of HB around veraison induced a higher color intensity in berries, and vines treated at higher doses reached this stage earlier than the un-treated controls. There was also a clear increase in both grape anthocyanin concentration and total soluble solids without having a negative impact on total yield. We therefore, confirm the role of HB as a universal natural stomatal closure compound and propose a new use for HB in viticulture as a ripening inducer, by accelerating anthocyanin accumulation., This research was funded by Grant INNVAL10/18/005 from the Agència Valenciana de la Innovació (Spain). C.P. was a recipient of a predoctoral contract of the Generalitat Valenciana (ACIF/2019/187). D.S.I. is supported by AEI-FEDER grant AGL2017-83738-C3-3-R.

The volatile esters of (Z)-3-hexenol with acetic, propionic, isobutyric, or butyric acids are synthesized by alcohol acyltransferases (AAT) in plants. These compounds are differentially emitted when tomato plants are efficiently resisting an infection with Pseudomonas syringae pv. tomato. We have studied the defensive role of these green leaf volatile (GLV) esters in the tomato response to bacterial infection, by analyzing the induction of resistance mediated by these GLVs and the phenotype upon bacterial infection of tomato plants impaired in their biosynthesis. We observed that treatments of plants with (Z)-3-hexenyl propionate (HP) and, to a greater extent with (Z)-3-hexenyl butyrate (HB), resulted in stomatal closure, PR gene induction and enhanced resistance to the bacteria. HB-mediated stomatal closure was also effective in several plant species belonging to Nicotiana, Arabidopsis, Medicago, Zea and Citrus genus, and both stomatal closure and resistance were induced in HB-treated NahG tomato plants, which are deficient in salicylic acid (SA) accumulation. Transgenic antisense AAT1 tomato plants, which displayed a reduction of ester emissions upon bacterial infection in leaves, exhibited a lower ratio of stomatal closure and were hyper-susceptible to bacterial infection. Our results confirm the role of GLV esters in plant immunity, uncovering a SA-independent effect of HB in stomatal defense. Moreover, we identified HB as a natural stomatal closure compound with potential agricultural applications.

[EN] The volatile esters of (Z)-3-hexenol with acetic, propionic, isobutyric, or butyric acids are synthesized by alcohol acyltransferases (AAT) in plants. These compounds are differentially emitted when tomato plants are efficiently resisting an infection with Pseudomonas syringae pv. tomato. We have studied the defensive role of these green leaf volatile (GLV) esters in the tomato response to bacterial infection, by analyzing the induction of resistance mediated by these GLVs and the phenotype upon bacterial infection of tomato plants impaired in their biosynthesis. We observed that treatments of plants with (Z)-3-hexenyl propionate (HP) and, to a greater extent with (Z)-3-hexenyl butyrate (HB), resulted in stomatal closure, PR gene induction and enhanced resistance to the bacteria. HB-mediated stomatal closure was also effective in several plant species belonging to Nicotiana, Arabidopsis, Medicago, Zea and Citrus genus, and both stomatal closure and resistance were induced in HB-treated NahG tomato plants, which are deficient in salicylic acid (SA) accumulation. Transgenic antisense AAT1 tomato plants, which displayed a reduction of ester emissions upon bacterial infection in leaves, exhibited a lower ratio of stomatal closure and were hyper-susceptible to bacterial infection. Our results confirm the role of GLV esters in plant immunity, uncovering a SA-independent effect of HB in stomatal defense. Moreover, we identified HB as a natural stomatal closure compound with potential agricultural applications., This work was funded by Grant AICO/2017/048 from the Generalitat Valenciana and by Grant INNVAL10/18/005 from the Agencia Valenciana de la Innovacio (Spain). We would like to thank the Metabolomics Service of the IBMCP (Valencia, Spain), especially to Teresa Caballero for her excellent technical support in the VOCs quantification. We also thank Eduardo Moya for technical assistance.

[EN] Volatile organic compounds (VOCs) emitted by plants are secondary metabolites that mediate the plant interaction with pathogens and herbivores. These compounds may perform direct defensive functions, i. e., acting as antioxidant, antibacterial, or antifungal agents, or indirectly by signaling the activation of the plant's defensive responses. Using a non-targeted GC-MS metabolomics approach, we identified the profile of the VOCs associated with the differential immune response of the Rio Grande tomato leaves infected with either virulent or avirulent strains of Pseudomonas syringae DC3000 pv. tomato. The VOC profile of the tomato leaves infected with avirulent bacteria is characterized by esters of (Z)-3-hexenol with acetic, propionic, isobutyric or butyric acids, and several hydroxylated monoterpenes, e. g., linalool, a -terpineol, and 4-terpineol, which defines the profile of an immunized plant response. In contrast, the same tomato cultivar infected with the virulent bacteria strain produced a VOC profile characterized by monoterpenes and SA derivatives. Interestingly, the differential VOCs emission correlated statistically with the induction of the genes involved in their biosynthetic pathway. Our results extend plant defense system knowledge and suggest the possibility for generating plants engineered to over-produce these VOCs as a complementary strategy for resistance., This work was funded by Grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness.

[EN] The importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many incompatible plant-pathogen interactions, but less is known about signalling in compatible interactions. In this type of interaction, tomato plants have been found to accumulate high levels of 2,5-dihydroxybenzoic acid (gentisic acid, GA), a metabolic derivative of SA. Exogenous GA treatments induce in tomato plants a set of PR proteins that differ from those induced by salicylic acid. While SA accumulates in tomato plants mainly as 2-O-beta-D-glucoside, GA has only been found as 5-O-beta-D-xyloside. To characterize this step of the GA signalling pathway further, the present work focuses on the study of the GA-conjugating activity in tomato plants. A gentisate glycosyltransferase (GAGT) cDNA has been isolated and overexpressed in Pichia pastoris, and GA-conjugating activity was confirmed by detecting the xylosylated GA. The purified plant protein is highly specific for GA, showing no activity toward many other phenolic compounds, including SA. In addition, it shows an outstanding selectivity for UDP-xylose as the sugar donor, which differentiates this enzyme from most glycosyltransferases. Both the GA-conjugating activity and the corresponding mRNA show a strong, rapid, and transient induction upon treatment of tomato plants with GA or SA. Furthermore, its expression is rapidly induced by compatible infections. However, neither the gene nor the activity seems to respond to incompatible infections or wounding. The unique properties of this new glycosyltransferase suggest a specific role in regulating the free GA levels in compatible plant-pathogen interactions., This work has been supported by the Spanish Ministry of Science and Innovation (grant numbers BMC2003-07837 and BFU2006-11546).

[EN] In the present work, we have looked for the nature of the phenylpropanoids biosynthesized during the plant-pathogen reaction of two systems, Cucumis sativus and Cucumis melo infected with either prunus necrotic ringspot virus (PNRSV) or melon necrotic spot virus (MNSV), respectively. An accumulation of p-coumaric, caffeic and/or ferulic acids was observed in infected plant extracts hydrolysed with P-glucosidase or esterase. Analysis of undigested samples by HPLC/ESI revealed that these compounds are mainly forming esters with glucose: 1-O-coumaroyl-beta-glucose, 1-O-caffeoyl-beta-glucose, and 1-O-fer-uloyl-beta-glucose. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), the second enzyme of the plant phenylpropanoid pathway, plays a pivotal role in the synthesis of these hydroxycinnamic acids. Thus, we have isolated and characterised a cDNA clone encoding this enzyme from PNRSV-infected cucumber, and a partial cDNA from MNSV-infected melon leaves. The deduced amino acid sequence revealed a notable degree of identity with homologous C4H enzymes from other plant species. In agreement with the induction of the phenylpropanoids presently described, it is reported that in cucumber and melon leaves, both viral infections studied induced C4H mRNA expression. A similar induction was observed for the first pherylpropanoid biosynthetic enzyme, phyenylalanine ammonia-lyase (PAL, EC 4.3.1.5), and for chitinase and peroxidase defence-related genes., We gratefully acknowledge Dr. Lynne Yenush for critical reading of the manuscript and helpful discussions, and L. Latorre, and L. Coracha´n for the technical support in the greenhouse. The authors want to acknowledge the detailed suggestions of the referees. This work was supported by Grant BMC2000-1136 from Comisio´n Interministerial de Ciencia y Tecnologı´a, Spanish Ministry of Science and Technology

[EN] Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants., This work was supported by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness received by JMB. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

[EN] We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent., This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnologicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.

[EN] Moringa oleifera seeds contain a cationic coagulant protein that can be used either for drinking water clarification or wastewater treatment. The seeds contain 30-40% (w/w) oil, which represents a valuable resource with industrial, food and renewable energy applications. However, the oil also increases the organic matter added to water and avoids storage of treated water and its consumption within 24h. So oil extraction is recommended as a way to purify the extract. This work proposes solvent extraction using three solvents (hexane, acetone, ethanol) and two procedures (batch and Soxhlet) and analyses for the first time the influence that these factors have on the coagulant efficiency of the extract prepared using defatted seeds. Results show that solvent-defatted seed extracts have a coagulant efficiency comparable to that observed for polyaluminium chloride coagulant (up to 88%), while non-defatted seed extract efficiency is 30% lower. Moreover, ethanol and Soxhlet method for oil extraction allows obtaining a defatted extract that requires doses of coagulant protein 5-33 times lower than hexane or acetone defatted extract to clarify turbid water, reducing in consequence the organic load added. SDS-PAGE analysis showed no influence of the solvent on the nature of the coagulant protein present in the extracts. The results of the study provide an oil extraction procedure that recovers M. oleifera oil for industrial purposes and enhances the defatted seeds obtaining an improved coagulant for drinking water treatment., The authors thank V. Conejero and A. Saurí from Instituto Mixto Universitario de Biología Molecular y Celular de Plantas (IBMCP UPV-CSIC), for their excellent technical assistance, and M.D. Climent, X. Climent and A. Sepulcre for their collaboration on this research. The authors wish to thank CCD-UPV for the financial support given to this research, through ADSI-DEO 2012 call and to Support Programme for Research and Development (PAID-06-12) from Universitat Politècnica de València

Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.

[EN] Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA: tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-over-expressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection., The authors are grateful to C. Torres, A. Sauri, and T. Caballero for technical support. We also thank C. Gomez-Mena for her assistance in tomato flower sampling. This work was supported by Grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, from the Spanish Ministry of Science and Innovation. L. Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). The work presented here was carried out in collaboration between all authors. J. M. Belles defined the research theme. L. Campos, L. Zacares and M. P. Lopez-Gresa carried out the laboratory experiments. P. Lison and I. Rodrigo contributed to the experimental design and the interpretation of the data. L. Campos drafted the article. M. P. Lopez-Gresa, P. Lison and I. Rodrigo participated in revising it critically for important intellectual content. J. M. Belles and V. Conejero gave the final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for the content.

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.

[EN] 1H nuclear magnetic resonance (NMR)-based metabolomics has been applied to study the compatible interaction between tomato plants and Tomato Mosaic Virus (ToMV). A detailed time course of metabolic fingerprinting of ToMV-inoculated and non-inoculated systemically infected tomato leaves has provided a fundamental understanding of the metabolic state of the plant not only in response to ToMV infection, but also under various physiological conditions. By this analytical platform a total of 32 metabolites including amino/organic acids, sugars, phenylpropanoids, flavonoids and other miscellaneous compounds were detected. Using multivariate data analysis, we have identified a subset of metabolites induced during the plant defence response and metabolites whose accumulation was dependent on the developmental stage, the position of the leaf on the stem, and the harvesting time. Specifically, a general time-dependent decrease in organic acids, amino acids (excluding asparagine), phenylpropanoids and rutin was observed in individual leaves. In addition, metabolite alterations were also found to correlate with the developmental stage of the leaf: high levels of organic acids, some amino acids, phenylpropanoids, and flavonoids were found in lower leaves while elevated amounts of sugars were present in the upper ones. Moreover, a marked variation in the content of some metabolites was also observed to be associated to the asymptomatic ToMV infection both in inoculated and systemically infected leaves. While flavonoids accumulated in virus-inoculated leaves, increased levels of phenylpropanoids were observed in non-inoculated leaves where ToMV actively replicates. Finally, diurnal changes in the metabolite content were also observed: an increase of amino acids and organic acids (except glutamic acid) were observed in the samples collected in the morning, whereas sugars and secondary metabolite levels increased in the tomato leaves harvested in the evening, The authors are grateful to Cristina Torres Vidal for technical support and Dr. Lynne Yenush for critical reading of the manuscript and exciting intellectual discussions. This work was supported by Grant BFU 2009-11958 from Secretaria de Estado de Investigacion from Spanish Ministry of Science and Innovation. M.P.L.G held a postdoctoral fellowship JAEDoc 08 00402 from the Consejo Superior de Investigaciones Cientificas (Spain) and was recipient of a postdoctoral fellowship (BEST/2010/111) from Generalitat Valencia (Spain).

[EN] In the present work, we aimed to define the chemical nature of the phenylpropanoids induced during the interaction of the tomato plant. cultivar Rutgers, with the bacterial pathogen Pseudomonas syringae pv. tomato. Using LC-MS and NMR spectroscopy, we determined the chemical structures of this class of metabolites, which accumulated in response to exposure to the pathogen. A rapid accumulation of phytoalexin hydroxycinnamic acid amides (HCAA) of noradrenaline (cis/trans N-p-coumaroylnoradrenaline and cis/trans N-feruloylnoradrenline) and octopamine (cis/trans N-p-coumaroyloctopamine and cis/trans N-feruloyloctopamine), chlorogenic acid (CGA), and the flavonoid rutin was detected after bacterial infiltration. Among them, cis N-p-coumaroylnoradrenaline, cis N-p-coumaroyloctopamine, and isomers cis/trans of N-feruloylnoradrenaline have never been described in the literature. Moreover, cis/trans isomers of N-feruloyloctopamine are identified for the first time as being associated with the tomato/P. syringae interaction. Inhibition of the P. syringae-induced ethylene by 2-aminoethoxyvinylglycine, markedly impaired the accumulation of HCAA in inoculated tomato leaves, but it had no effect on CGA or rutin synthesis. On the other hand, the lack of accumulation of SA in NahG transgenic tomato plants, overexpressing a bacterial salicylic hydroxylase, did not prevent the enhancing of HCAA produced by P. syringae infection. Taken together, our results indicate that ethylene, but not SA, is essential for the synthesis of HCAA in response to bacterial infection of tomato leaves. Antibiotic activities of the induced metabolites were also studied. CGA induced a notable expression of the defence-related genes PR1 and P23. Moreover, trans-HCAA of noradrenaline and octopamine showed a potent free radical scavenging competence. In particular, trans N-feruloylnoradrenaline presented a very outstanding antioxidant activity, thus indicating that these compounds may play a role in the defence response of tomato plants against bacterial infection. (C) 2011 Elsevier B.V. All rights reserved., The authors want to thank Dr. Lynne Yenush for critical reading of the manuscript and exciting intellectual discussions. Dominique Melck, Estrella Mateos (ICB and ITQ NMR Service) for recording NMR spectra and Letizia Ciavatta for interpretation of the 600-MHz NMR spectra. This work was supported by Grant BFU2006-11546 from Direccion General de Programas y Transferencia de Conocimiento, from Spanish Ministry of Science and Innovation. M.P.L.G. was the recipient of a postdoctoral fellowship APOSTD/2007/128 from Generalitat Valencia (Spain).

P23, a 23 kDa pathogenesis-related (PR) protein, was purified from citrus exocortis viroid (CEVd)-infected tomato leaves. Partial amino acid sequencing of this protein including the N-terminal and nine additional tryptic fragments covering about 50% of its primary structure revealed extensive homologies to the members of the family of plant thaumatin-like proteins. Sequence alignment revealed that tomato P23 is the previously described NP24 protein found to be associated to osmotic stress in tomato. In view of this fact the possible role of pathogenesis-related P23 protein as a component of a general mechanism of response of the plant is discussed.

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (beta-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Belles et al. in Mol Plant-Microbe Interact 12:227-235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Despite the fact that viroids are the infectious agents with the lowest complexity and best known structure, our understanding of their pathogenic interaction with the host plant is still far from complete. Viroid pathogenesis poses many intriguing questions; these have to be answered without any viroid-specified protein to which to attribute a pathogenic role. Here we present a view of viroid pathogenesis in which viroid molecules are considered as pure replicating and pathogenic signals. These act as elicitors of host responses which can also be activated by other afflicting agents. A model is outlined to explain how the viroid-induced response at the cellular level becomes a developmental disease and leads to a plant which is more resistant to subsequent infections.

As a response to the stress induced by different afflicting agents, tomato plants (Lycopersicon esculentum) produce the so-called 'pathogenesis-related' proteins. Here we report the degradation of some of these proteins by a constitutive endoproteolytic activity that co-distributes with pathogenesis-related proteins in the intercellular spaces of tomato leaves infected with citrus exocortis viroid. This endoproteinase was purified, showing a pH optimum of 2.5-3.5, a Mr of 37,000 and selective inhibition by pepstatin. In crude homogenates, the enzyme does not seem to degrade other cellular proteins. This specificity indicates that the proteinase might be involved in the extracellular degradative pathway of pathogenesis-related proteins and in the regulation of their biological function.

A 23-kD pathogenesis-related protein (P23) is induced in tomato (Lycopersicon esculentum Mill, cv Rutgers) plants when infected with citrus exocortis viroid. This protein is homologous to the salt-induced tomato NP24 protein (I. Rodrigo, P. Vera, R. Frank, V. Conejero [1991] Plant Mol Biol 16: 931–)934). Further characterization of P23 has shown that this protein accumulates in vacuoles in association with dense inclusion bodies. In vitro assays indicated that the purified P23 protein inhibits the growth of several phytopathogenic fungi. P23-coding cDNA clones were isolated from viroid-induced and ethylene-induced libraries. Southern analysis showed that at least two genes could encode P23 or P23-related products. The accumulation of P23 protein correlated with the accumulation of its mRNA. Sequence analysis revealed significant differences in both coding and downstream untranslated regions between the cDNA sequences corresponding to the viroid-induced P23 and the salt stress-induced NP24 proteins.

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.