Eliane Abou-Mansour

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus‐induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate‐derived synthesis of SA in N. benthamiana

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.

Disease resistance of Arabidopsis to Phytophthora brassicae is established by the sequential action of indole glucosinolates and camalexin.

We have analysed the role of tryptophan-derived secondary metabolites in disease resistance of Arabidopsis to the oomycete pathogen Phytophthora brassicae. Transcript analysis revealed that genes encoding enzymes involved in tryptophan, camalexin and indole glucosinolate (iGS) biosynthesis are coordinately induced in response to P. brassicae. However, a deficiency in either camalexin or iGS accumulation has only a minor effect on the disease resistance of Arabidopsis mutants. In contrast, the double mutant cyp79B2 cyp79B3, which has a blockage in the production of indole-3-aldoxime (IAOx), the common precursor of tryptophan-derived metabolites including camalexin and iGS, is highly susceptible to P. brassicae. Because cyp79B2 cyp79B3 shows no deficiencies in other tested disease resistance responses, we concluded that the lack of IAOx-derived compounds renders Arabidopsis susceptible despite wild-type-like pathogen-induced hypersensitive cell death, stress hormone signaling and callose deposition. The susceptibility of the double mutant pen2-1 pad3-1, which has a combined defect in camalexin synthesis and PEN2-catalysed hydrolysis of iGS compounds, demonstrates that both camalexin and products of iGS hydrolysis are important for disease resistance to P. brassicae. Products of iGS hydrolysis play an early defensive role, as indicated by enhanced epidermal penetration rates of Arabidopsis mutants affected in iGS synthesis or degradation. Our results show that disease resistance of Arabidopsis to P. brassicae is established by the sequential activity of the phytoanticipin iGS and the phytoalexin camalexin.

Export of salicylic acid from the chloroplast requires the multidrug and toxin extrusion-like transporter EDS5.

The synthesis of the immune signal salicylic acid is abolished by a mutation in a hitherto unknown transporter protein. This article describes the transporter localization at the chloroplast and its function in the export of salicylic acid from the chloroplast. Salicylic acid (SA) is central for the defense of plants to pathogens and abiotic stress. SA is synthesized in chloroplasts from chorismic acid by an isochorismate synthase (ICS1); SA biosynthesis is negatively regulated by autoinhibitory feedback at ICS1. Genetic studies indicated that the multidrug and toxin extrusion transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) of Arabidopsis (Arabidopsis thaliana) is necessary for SA accumulation after biotic and abiotic stress, but so far it is not understood how EDS5 controls the biosynthesis of SA. Here, we show that EDS5 colocalizes with a marker of the chloroplast envelope and that EDS5 functions as a multidrug and toxin extrusion-like transporter in the export of SA from the chloroplast to the cytoplasm in Arabidopsis, where it controls the innate immune response. The location at the chloroplast envelope supports a model of the effect of EDS5 on SA biosynthesis: in the eds5 mutant, stress-induced SA is trapped in the chloroplast and inhibits its own accumulation by autoinhibitory feedback.

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2 −, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

Salicylic acid and its location in response to biotic and abiotic stress

Salicylic acid (SA) is an important signal involved in the activation of plant defence responses against abiotic and biotic stress. SA may derive from the phenylpropanoid pathway or via isochorismate synthase as demonstrated in Nicotiana benthamiana, tomato and Arabidopsis thaliana. The phenylpropanoid pathway as well as isochorismate synthase are localized in the chloroplasts but it remains unknown if the end product SA is in the same organelle. We have studied the localization of SA in A. thaliana using the salicylate hydroxylase (NahG) gene expressed with a chloroplast targeting sequence. Plants expressing NahG in the chloroplasts are unable to accumulate SA induced after pathogen or UV exposure. Our data infer that SA is initially located in the chloroplasts.

Perception of soft mechanical stress in Arabidopsis leaves activates disease resistance

BackgroundIn a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound–induced resistance to B. cinerea.ResultsIn this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves.ConclusionArabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.

Identification of phytotoxins from Botryosphaeria obtusa , a pathogen of black dead arm disease of grapevine

A bioassay-guided fractionation of a culture filtrate of Botryosphaeria obtusa led to the isolation of four dihydroisocoumarins, named mellein 1, 4-hydroxymellein 2, 7-hydroxymellein 3 and the new 4,7-dihydroxymellein 4. LC-UV-DAD-MS analysis of vine wood infected by B. obtusa revealed the presence of mellein (1). Botryosphaeria obtusa was also able to oxidise wood δ-resveratrol into the dimer delta-viniferin. The structures of isolated phytotoxins were established on the basis of IR, MS, 1D and 2D NMR.

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine.

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, (-)-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) (-)-4-chloro-terremutin hydrate (4) and(+)-4-hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-(-)-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)(-)-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with (-)-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxins.

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity.In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed.Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed.