Plant triterpenoid saponins function as susceptibility factors to promote the pathogenicity of Botrytis cinerea.

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen that causes diseases in hundreds of plant species, including high-value crops. Its polyxenous nature and pathogenic success are due to its ability to perceive host signals in its favor. In this study, we found that laticifer cells of Euphorbia lathyris are a source of susceptibility factors required by B. cinerea to cause disease. Consequently, poor-in-latex (pil) mutants, which lack laticifer cells, show full resistance to this pathogen, whereas lot-of-latex mutants, which produce more laticifer cells, are hypersusceptible. These S factors are triterpenoid saponins, which are widely distributed natural products of vast structural diversity. The downregulation of laticifer-specific oxydosqualene cyclase genes, which encode the first committed step enzymes for triterpene and, therefore, saponin biosynthesis, conferred disease resistance to B. cinerea. Likewise, the Medicago truncatula lha-1 mutant, compromised in triterpenoid saponin biosynthesis, showed enhanced resistance. Interestingly, the application of different purified triterpenoid saponins pharmacologically complemented the disease-resistant phenotype of pil and hla-1 mutants and enhanced disease susceptibility in different plant species. We found that triterpenoid saponins function as plant cues that signal transcriptional reprogramming in B. cinerea, leading to a change in its growth habit and infection strategy, culminating in the abundant formation of infection cushions, the multicellular appressoria apparatus dedicated to plant penetration and biomass destruction in B. cinerea. Taken together, these results provide an explanation for how plant triterpenoid saponins function as disease susceptibility factors to promote B. cinerea pathogenicity.

Metabolic crosstalk between hydroxylated monoterpenes and salicylic acid in tomato defense response against bacteria.

Hydroxylated monoterpenes (HMTPs) are differentially emitted by tomato (Solanum lycopersicum) plants resisting bacterial infection. We have studied the defensive role of these volatiles in the tomato response to bacteria, whose main entrance is through stomatal apertures. Treatments with some HMTPs resulted in stomatal closure and pathogenesis-related protein 1 (PR1) induction. Particularly, α-terpineol induced stomatal closure in a salicylic acid (SA) and abscisic acid-independent manner and conferred resistance to bacteria. Interestingly, transgenic tomato plants overexpressing or silencing the monoterpene synthase MTS1, which displayed alterations in the emission of HMTPs, exhibited changes in the stomatal aperture but not in plant resistance. Measures of both 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) and SA levels revealed competition for MEcPP by the methylerythritol phosphate (MEP) pathway and SA biosynthesis activation, thus explaining the absence of resistance in transgenic plants. These results were confirmed by chemical inhibition of the MEP pathway, which alters MEcPP levels. Treatments with benzothiadiazole (BTH), a SA functional analog, conferred enhanced resistance to transgenic tomato plants overexpressing MTS1. Additionally, these MTS1 overexpressors induced PR1 gene expression and stomatal closure in neighboring plants. Our results confirm the role of HMTPs in both intra- and interplant immune signaling and reveal a metabolic crosstalk between the MEP and SA pathways in tomato plants.

Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure.

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators.

Transcriptional regulators based on CRISPR architecture expand our ability to reprogramme endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely, naringenin, eriodictyol, kaempferol, and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programmes for each of the four metabolites, each programme activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation programme showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples into five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for the custom-made design of metabolic contents in plants.

Untargeted Metabolomics of Rind Essential Oils Allowed to Differentiate Two Closely Related Clementine Varieties.

Chemical characterization of clementine varieties (Citrus clementina Hort. ex Tan.) essential oils (EO) can lead to variety identification and valorization of their potential use in food and aroma industries. The goal of this study was the chemometric discrimination between two very closely related and morphologically identical clementine varieties, Clemenules (NL) and Clemenpons (PO), based on their rind EO, to identify the differential volatile organic compounds (VOCs) and to determine their antioxidant capacity. EO rind volatile profile was determined by gas chromatography coupled to mass spectrometry in Citrus fruit at different ripening stages grown two independent years in two different locations. Untargeted metabolomics and multivariate data analysis showed an evolution of EO volatile profiles markedly parallel in both varieties. Although EO qualitative composition was identical in both varieties, PLS-DA allowed the identification of characteristic VOCs, quantitatively discriminating them along all the ripening process. PO showed higher accumulation of several mono- and sesquiterpene compounds such as trans-carveol, while NL showed higher levels of aldehyde and alcohol non-terpenoids like dodecanal. Both varieties evinced identical EO antioxidant activities, indicating a similar value for food preservation. Hence, untargeted metabolomics approach based on rind EO volatiles was revealed as a powerful technique able to differentiate between morphologically undistinguishable Citrus varieties.

Symptom Severity, Infection Progression and Plant Responses in Solanum Plants Caused by Three Pospiviroids Vary with the Inoculation Procedure.

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.

Tomato glycosyltransferase Twi1 plays a role in flavonoid glycosylation and defence against virus.

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.

Conservation of Thermospermine Synthase Activity in Vascular and Non-vascular Plants.

In plants, the only confirmed function for thermospermine is regulating xylem cells maturation. However, genes putatively encoding thermospermine synthases have been identified in the genomes of both vascular and non-vascular plants. Here, we verify the activity of the thermospermine synthase genes and the presence of thermospermine in vascular and non-vascular land plants as well as in the aquatic plant Chlamydomonas reinhardtii. In addition, we provide information about differential content of thermospermine in diverse organs at different developmental stages in some vascular species that suggest that, although the major role of thermospermine in vascular plants is likely to be xylem development, other potential roles in development and/or responses to stress conditions could be associated to such polyamine. In summary, our results in vascular and non-vascular species indicate that the capacity to synthesize thermospermine is conserved throughout the entire plant kingdom.

Effect of Benzothiadiazole on the Metabolome of Tomato Plants Infected by Citrus Exocortis Viroid.

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.

A New Role For Green Leaf Volatile Esters in Tomato Stomatal Defense Against Pseudomonas syringe pv. tomato.

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.

Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs.

N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

Drought Tolerance in Pinus halepensis Seed Sources As Identified by Distinctive Physiological and Molecular Markers.

Drought is one of the main constraints determining forest species growth, survival and productivity, and therefore one of the main limitations for reforestation or afforestation. The aim of this study is to characterize the drought response at the physiological and molecular level of different Pinus halepensis (common name Aleppo pine) seed sources, previously characterized in field trials as drought-sensitive or drought-tolerant. This approach aims to identify different traits capable of predicting the ability of formerly uncharacterized seedlings to cope with drought stress. Gas-exchange, water potential, photosynthetic pigments, soluble sugars, free amino acids, glutathione and proteomic analyses were carried out on control and drought-stressed seedlings in greenhouse conditions. Gas-exchange determinations were also assessed in field-planted seedlings in order to validate the greenhouse experimental conditions. Drought-tolerant seed sources presented higher values of photosynthetic rates, water use efficiency, photosynthetic pigments and soluble carbohydrates concentrations. We observed the same pattern of variation of photosynthesis rate and maximal efficiency of PSII in field. Interestingly drought-tolerant seed sources exhibited increased levels of glutathione, methionine and cysteine. The proteomic profile of drought tolerant seedlings identified two heat shock proteins and an enzyme related to methionine biosynthesis that were not present in drought sensitive seedlings, pointing to the synthesis of sulfur amino acids as a limiting factor for drought tolerance in Pinus halepensis. Our results established physiological and molecular traits useful as distinctive markers to predict drought tolerance in Pinus halepensis provenances that could be reliably used in reforestation programs in drought prone areas.

A Non-targeted Metabolomics Approach Unravels the VOCs Associated with the Tomato Immune Response against Pseudomonas syringae.

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, α-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.

Transgenic tomato plants overexpressing tyramine N-hydroxycinnamoyltransferase exhibit elevated hydroxycinnamic acid amide levels and enhanced resistance to Pseudomonas syringae.

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-overexpressing 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.

Two copper complexes from two novel naphthalene-sulfonyl-triazole ligands: different nuclearity and different DNA binding and cleavage capabilities.

Two novel naphthalene-sulfonyl-triazole ligands, 5-amino-N1-(naphthalen-3-ylsulfonyl)-1,2,4-triazole (anstrz) and 3,5-diamino-N1-(naphthalen-3-ylsulfonyl)-1,2,4-triazole (danstrz), purposely designed to interact with DNA, have been prepared for the first time and then fully characterized by (1)H, (13)C NMR, and IR spectroscopy, mass spectrometry and elemental analysis. The crystal structures of two copper complexes of these derivatives, i.e. [Cu(anstrz)4(NO3)2]∙4CH3OH (1), mononuclear, and [Cu(danstrz)(µ-OAc)2]2∙2(danstrz) (OAc=acetato) (2), dinuclear, have been determined by single-crystal X-ray diffraction. In both cases the ligand coordinates in a monodentate fashion via the N4 nitrogen atom of the triazole ring. Compound 2, an example of paddle wheel type copper acetate, presents a Cu⋯Cu' distance of 2.667(1) Å. As a result of strong stacking interactions and intense H-bonds, the structure of 2 constitutes a MOF (metal-organic framework). Besides, this dinuclear compound exhibits a very strong antiferromagnetic coupling (J=-324cm(-1)) and a silent X-band EPR at room temperature. The affinity toward DNA of 1 and 2 has been examined by fluorescence emission spectroscopy, thermal denaturation and viscosimetry assays. The apparent binding constant (Kapp) values of 2.2×10(7)M(-1) for 1 and 2.6×10(7)M(-1) for 2 suggest important DNA interaction. The dinuclear compound (2) intercalates and produces a high change in the Tm. Both compounds promote DNA scission in the presence of H2O2/ascorbate (1) or ascorbate (2) through oxidative mechanism. The possible reasons for the higher DNA affinity and the more efficient DNA cleavage displayed by compound 2 in relation to compound 1 are discussed.

The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway.

Fruit-set and growth in tomato depend on the action of gibberellins (GAs). To evaluate the role of the GA biosynthetic enzyme GA 20-oxidase (GA20ox) in that process, the citrus gene CcGA20ox1 was overexpressed in tomato (Solanum lycopersicum L.) cv Micro-Tom. The transformed plants were taller, had non-serrated leaves, and some flowers displayed a protruding stigma due to a longer style, thus preventing self-pollination, similar to GA(3)-treated plants. Flowering was delayed compared with wild-type (WT) plants. Both yield and number of fruits per plant, some of them seedless, were higher in the transgenic plants. The Brix index value of fruit juice was also higher due to elevated citric acid content, but not glucose or fructose content. When emasculated, 14-30% of ovaries from transgenic flowers developed parthenocarpically, whereas no parthenocarpy was found in emasculated WT flowers. The presence of early-13-hydroxylation and non-13-hydroxylation GA pathways was demonstrated in the shoot and fruit of Micro-Tom, as well as in two tall tomato cultivars (Ailsa Craig and UC-82). The transgenic plants had altered GA profiles containing higher concentrations of GA(4), from the non-13-hydroxylation pathway, which is generally a minor active GA in tomato. The effect of GA(4) application in enhancing stem growth and parthenocarpic fruit development was proportional to dose, with the same activity as GA(1). The results support the contention that GA20ox overexpression diverts GA metabolism from the early-13-hydroxylation pathway to the non-13-hydroxylation pathway. This led to enhanced GA(4) synthesis and higher yield, although the increase in GA(4) content in the ovary was not sufficient to induce full parthenocarpy.

Metabolic characterization of Withania somnifera from different regions of India using NMR spectroscopy.

Withania somnifera (L.) Dun. (Solanaceae), known as Indian ginseng, is one of the most popular medicinal plants in India. Considering the importance and common use of this plant, it is necessary to investigate its holistic metabolite profile. However, with existing analytical methods which are based on TLC and HPLC­UV (or MS), it is difficult to obtain information of the whole range of compounds appropriately. In this study, the metabolic characterization of Withania somnifera leaves, stems, and roots collected in six different regions in India was performed using ¹H NMR spectroscopy followed by principal component analysis (PCA) and hierarchical clustering analysis (HCA). Of the parts of Withania somnifera analyzed in this study, the leaf was found to have the widest range of metabolites, including amino acids, flavonoids, lipids, organic acids, phenylpropanoids, and sugars, as well as the main secondary metabolites of the plant, withanolides. The ¹H NMR spectra revealed the presence of two groups of withanolides: 4-OH and 5,6-epoxy withanolides (withaferin A-like steroids) and 5- OH and 6,7-epoxy withanolides (withanolides Alike steroids). The ratio of these two withanolides was found to be a key discriminating feature of Withania somnifera leaf samples from different origins.

Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid.

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-ß-D-glucoside, GA has only been found as 5-O-ß-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.

Induction of p-coumaroyldopamine and feruloyldopamine, two novel metabolites, in tomato by the bacterial pathogen Pseudomonas syringae.

Inoculation of tomato plants (Solanum lycopersicum cv. Rutgers) with Pseudomonas syringae pv. tomato led to the production of a hypersensitive-like response in this pathovar of tomato. Accumulation of hydroxycinnamic acid amides (HCAA) of tyramine (p-coumaroyltyramine and feruloyltyramine) and dopamine (p-coumaroyldopamine and feruloyldopamine) was detected after bacterial infection. Two of them, p-coumaroyldopamine and feruloyldopamine, are described for the first time. The accumulation of HCAA was preceded by an increment of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) gene expression. HCAA also accumulated in transgenic NahG tomato plants overexpressing a bacterial salicylic hydroxylase. However, treatment of plants with the ethylene biosynthesis inhibitor, aminoethoxyvinilglycine, led to a reduction in the accumulation of THT transcripts and HCAA. Together, the results suggest that pathogen-induced induction of ethylene is essential for HCAA synthesis, whereas salicylic acid is not required for this response. In addition, notable antibacterial and antioxidant activities were found for the new HCAA, thus indicating that they could play a role in the defense of tomato plants against bacterial infection.

Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens.

Production of fungitoxic extrolites was evaluated in culture filtrates of several isolates belonging to Penicillium canescens and P. janczewskii that showed some extent of inhibitory activity against the plant pathogenic fungus Rhizoctonia solani. In addition to griseofulvin and dechlorogriseofulvin that are already known in these species, curvulinic acid, previously unreported in Penicillium, was produced by all isolates assayed. Another extrolite recently characterized from a P. verrucosum strain by the name of Sch 642305 was detected in 5 isolates of P. canescens only. The purified compound completely inhibited mycelial growth of isolates of Rhizoctonia solani and other plant pathogenic fungi in vitro. The role of this extrolite as a possible biochemical determinant of antagonism toward plant pathogenic fungi, and implications concerning chemotaxonomy are discussed.