Showing papers on "Phytoalexin published in 2015"

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance.

Abstract: Maize (Zea mays) production, which is of global agro-economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought-induced phytoalexins is positively correlated with the root-to-shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.

Maize death acids, 9-lipoxygenase–derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators

Abstract: Plant damage promotes the interaction of lipoxygenases (LOXs) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides, and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed "jasmonates." As signals, jasmonates have related yet distinct roles in the regulation of plant resistance against insect and pathogen attack. A similar pathway involving 9-LOX activity on linolenic and linoleic acid leads to the 12-OPDA positional isomer, 10-oxo-11-phytodienoic acid (10-OPDA) and 10-oxo-11-phytoenoic acid (10-OPEA), respectively; however, physiological roles for 9-LOX cyclopentenones have remained unclear. In developing maize (Zea mays) leaves, southern leaf blight (Cochliobolus heterostrophus) infection results in dying necrotic tissue and the localized accumulation of 10-OPEA, 10-OPDA, and a series of related 14- and 12-carbon metabolites, collectively termed "death acids." 10-OPEA accumulation becomes wound inducible within fungal-infected tissues and at physiologically relevant concentrations acts as a phytoalexin by suppressing the growth of fungi and herbivores including Aspergillus flavus, Fusarium verticillioides, and Helicoverpa zea. Unlike previously established maize phytoalexins, 10-OPEA and 10-OPDA display significant phytotoxicity. Both 12-OPDA and 10-OPEA promote the transcription of defense genes encoding glutathione S transferases, cytochrome P450s, and pathogenesis-related proteins. In contrast, 10-OPEA only weakly promotes the accumulation of multiple protease inhibitor transcripts. Consistent with a role in dying tissue, 10-OPEA application promotes cysteine protease activation and cell death, which is inhibited by overexpression of the cysteine protease inhibitor maize cystatin-9. Unlike jasmonates, functions for 10-OPEA and associated death acids are consistent with specialized roles in local defense reactions.

Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae

Abstract: Summary Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2O2), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling.

Phenolic Phytoalexins in Rice: Biological Functions and Biosynthesis

Abstract: Phytoalexins are inducible secondary metabolites possessing antimicrobial activity against phytopathogens. Rice produces a wide array of phytoalexins in response to pathogen attacks and environmental stresses. With few exceptions, most phytoalexins identified in rice are diterpenoid compounds. Until very recently, flavonoid sakuranetin was the only known phenolic phytoalexin in rice. However, recent studies have shown that phenylamides are involved in defense against pathogen attacks in rice. Phenylamides are amine-conjugated phenolic acids that are induced by pathogen infections and abiotic stresses including ultra violet (UV) radiation in rice. Stress-induced phenylamides, such as N-trans-cinnamoyltryptamine, N-p-coumaroylserotonin and N-cinnamoyltyramine, have been reported to possess antimicrobial activities against rice bacterial and fungal pathogens, an indication of their direct inhibitory roles against invading pathogens. This finding suggests that phenylamides act as phytoalexins in rice and belong to phenolic phytoalexins along with sakuranetin. Phenylamides also have been implicated in cell wall reinforcement for disease resistance and allelopathy of rice. Synthesis of phenolic phytoalexins is stimulated by phytopathogen attacks and abiotic challenges including UV radiation. Accumulating evidence has demonstrated that biosynthetic pathways including the shikimate, phenylpropanoid and arylmonoamine pathways are coordinately activated for phenolic phytoalexin synthesis, and related genes are induced by biotic and abiotic stresses in rice.

Elicitation and accumulation of stilbene phytoalexins in grapevine berries infected by Botrytis cinerea

Abstract: At three developmental stages berries of field-grown Castor (interspecific crossing) and Huxelrebe ( V vinifera L crossing) were in vitro inoculated with two strains of Botrytis cinerea Pers to investigate the response of berries to fungal infection With respect to the time course of phytoalexin (trans-resveratrol, e-viniferin and pterostilbene) accumulation and Visual disease symptoms In infected berries the amounts of e-viniferin dominated over pterostilbene The stilbene phytoalexin content decreased during berry development and sugar accumulation Grape varieties reacted differently to B cinerea strains with regard to stilbene response and Visual symptoms Mechanical damage of the berry skin induced uninfected berries to synthesize low amounts of phytoalexins It can be assumed that after extraction and degradation e-viniferin of mature berries is a source of resveratrol in wine

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

Abstract: Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

Jasmonic Acid, Abscisic Acid, and Salicylic Acid Are Involved in the Phytoalexin Responses of Rice to Fusarium fujikuroi, a High Gibberellin Producer Pathogen.

Abstract: Fusarium fujikuroi, the causal agent of bakanae disease, is the main seedborne pathogen on rice To understand the basis of rice resistance, a quantitative method to simultaneously detect phytohormones and phytoalexins was developed by using HPLC–MS/MS With this method dynamic profiles and possible interactions of defense-related phytohormones and phytoalexins were investigated on two rice cultivars, inoculated or not with F fujikuroi In the resistant cultivar Selenio, the presence of pathogen induced high production of phytoalexins, mainly sakuranetin, and symptoms of bakanae were not observed On the contrary, in the susceptible genotype Dorella, the pathogen induced the production of gibberellin and abscisic acid and inhibited jasmonic acid production, phytoalexins were very low, and bakanae symptoms were observed The results suggested that a wide range of secondary metabolites are involved in plant defense against pathogens and phytoalexin synthesis could be an important factor for rice resistance

Analysis of the Molecular Dialogue Between Gray Mold (Botrytis cinerea) and Grapevine (Vitis vinifera) Reveals a Clear Shift in Defense Mechanisms During Berry Ripening.

Abstract: 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 potential relationship of stilbene (resveratrol) synthesis to anthocyanin content in grape berry skins

Abstract: The relationship between the production of resveratrol, a phytoalexin related to grape disease resistance, and the anthocyanin content of grape berries in diverse Vitis species has been investigated. Previous studies have reported that the phytoalexin production potential of grapes suddenly declines at veraison. The results obtained here from assaying resveratrol and anthocyanins from grape berries in different developmental stages suggest that chalcone synthase (EC 2.3.1.74), the key enzyme involved in anthocyanin biosynthesis, may compete with stilbene (resveratrol) synthase (EC 2.3.1.-), such that the decrease of the ability of grapes to synthesize resveratrol in response to UV-irradiation observed after the onset of fruit ripening may be a consequence of the concomitant rise of anthocyanin accumulation in fruits.

Deep sequencing analysis of transcriptomes in Aspergillus flavus in response to resveratrol

Abstract: Resveratrol has been reported as a natural phytoalexin that inhibits infection or the growth of certain fungi including Aspergillus flavus. Our previous research revealed that aflatoxin production in A. flavus was reduced in medium with resveratrol. To understand the molecular mechanism of the A. flavus response to resveratrol treatment, the high-throughput paired-end RNA-Seq was applied to analyze the transcriptomic profiles of A. flavus. In total, 366 and 87 genes of A. flavus were significantly up- and down- regulated, respectively, when the fungus was treated with resveratrol. Gene Ontology (GO) functional enrichment analysis revealed that 48 significantly differentially expressed genes were involved in 6 different terms. Most genes in the aflatoxin biosynthetic pathway genes cluster (#54) did not show a significant change when A. flavus was treated with resveratrol, but 23 of the 30 genes in the #54 cluster were down-regulated. The transcription of aflA and aflB was significantly suppressed under resveratrol treatment, resulting in an insufficient amount of the starter unit hexanoate for aflatoxin biosynthesis. In addition, resveratrol significantly increased the activity of antioxidative enzymes that destroy radicals, leading to decreased aflatoxin production. Moreover, stuA, fluG, flbC, and others genes involved in mycelial and conidial development were down-regulated, which disrupted the cell’s orderly differentiation and blocked conidia formation and mycelia development. The transcripts of laeA and veA were slightly inhibited by resveratrol, which may partly decrease aflatoxin production and depress conidia formation. Resveratrol can affect the expression of A. flavus genes that are related to developmental and secondary metabolic processes, resulting in decreased aflatoxin production and conidia formation and could also cause abnormal mycelia development. These results provide insight into the transcriptome of A. flavus in response to resveratrol and a new clew for further study in regulation of aflatoxin biosynthesis in A. flavus.

Identification of UV-Induced Diterpenes Including a New Diterpene Phytoalexin, Phytocassane F, from Rice Leaves by Complementary GC/MS and LC/MS Approaches.

Abstract: Rice phytoalexins are regarded as one of the most important weapons against pathogenic microorganisms. We attempted to identify novel phytoalexins and their derivatives using GC/MS and LC/MS analyses. Diterpene derivatives, 9β-pimara-7,15-diene-3β,6β,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, were isolated from UV-irradiated rice leaves by chromatographic methods. These structures were confirmed by 1D- and 2D-NMR and MS analyses. Interestingly, all three compounds were accumulated following an infection by the rice blast pathogen Magnaporthe oryzae. Compounds 1 and 2 exhibited weak antifungal activity and may be the biosynthetic intermediates of rice phytoalexins momilactones and oryzalexin S, respectively. Compound 3 exhibited relatively high inhibitory activity against the fungal mycelial growth of M. oryzae to the same extent as the known phytoalexin phytocassane A. We conclude that 3 is a member of the cassane-type phytoalexin family and propose the name phytocassane F.

Sustained exposure to abscisic acid enhances the colonization potential of the mutualist fungus Piriformospora indica on Arabidopsis thaliana roots

Abstract: Summary Root colonization by the beneficial fungus Piriformospora indica is controlled by plant innate immunity, but factors that channel this interaction into a mutualistic relationship are not known. We have explored the impact of abscisic acid (ABA) and osmotic stress on the P. indica interaction with Arabidopsis thaliana. The activation of plant innate immunity in roots was determined by measuring the concentration of the phytoalexin camalexin and expression of transcription factors regulating the biosynthesis of tryptophan-related defence metabolites. Furthermore, the impact of the fungus on the content of ABA, salicylic acid, jasmonic acid (JA) and JA-related metabolites was examined. We demonstrated that treatment with exogenous ABA or the ABA analogue pyrabactin increased fungal colonization efficiency without impairment of plant fitness. Concomitantly, ABA-deficient mutants of A. thaliana (aba1-6 and aba2-1) were less colonized, while plants exposed to moderate stress were more colonized than corresponding controls. Sustained exposure to ABA attenuated expression of transcription factors MYB51, MYB122 and WRKY33 in roots upon P. indica challenge or chitin treatment, and prevented an increase in camalexin content. The results indicate that ABA can strengthen the interaction with P. indica as a consequence of its impact on plant innate immunity. Consequently, ABA will be relevant for the establishment and outcome of the symbiosis under stress conditions.

A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X.

Abstract: Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

Resveratrol Targets in Inflammation.

Abstract: Resveratrol, a constituent of grapes and various other plants, has been an attractive compound for biomedical studies because moderate long-term drinking of red wine is associated with a reduced risk of lifestyle-related diseases, such as cardiovascular diseases and cancer. Resveratrol is as a phytoalexin, cyclooxygenase (COX) suppressor, and an activator of peroxisome proliferatoractivated receptor (PPAR) and SIRT1. As a major phytoalexin, resveratrol is produced by plants in response to various environmental stresses, such as pathogens and ultraviolet (UV) radiation, and promotes resistance to these stresses. A similar active ingredient, salicylic acid (SA), is also produced by plants. Aspirin, acetylated SA, is a major nonsteroidal anti-inflammatory drug (NSAID) because it inhibits COX activity in humans. The jasmonic acid (JA) pathway in plants and the COX pathway in humans are both defense systems against environmental stresses and involve lipid mediators derived from phospholipids. We can hypothesize that there is a molecular basis for the mutually beneficial relationship between plants and humans, which is important for understanding the mode of action of resveratrol in inflammation. Here we provide a review of the studies on resveratrol, especially with respect to the role of COX and PPAR in inflammation.

BTH and BABA induce resistance in pea against rust (Uromyces pisi) involving differential phytoalexin accumulation

Abstract: Systemic acquired resistance elicitors, BTH and BABA, reduce rust penetration in pea through phytoalexins pathway but differing in their mode of action. It has been previously shown that rust (Uromyces pisi) infection can be reduced in pea (Pisum sativum) by exogenous applications of systemic acquired resistance elicitors such as BTH and BABA. This protection is known to be related with the induction of the phenolic pathway but the particular metabolites involved have not been determined yet. In this work, we tackled the changes induced in phytoalexin content by BTH and BABA treatments in the context of the resistance responses to pea rust. Detailed analysis through high-performance liquid chromatography (HPLC) showed qualitative and quantitative differences in the content, as well as in the distribution of phytoalexins. Thus, following BTH treatment, we observed an increase in scopoletin, pisatin and medicarpin contents in all, excreted, soluble and cell wall-bound fraction. This suggests fungal growth impairment by both direct toxic effect as well as plant cell wall reinforcement. The response mediated by BTH was genotype-dependent, since coumarin accumulation was observed only in the resistant genotype whereas treatment by BABA primed phytoalexin accumulation in both genotypes equally. Exogenous application to the leaves of scopoletin, medicarpin and pisatin lead to a reduction of the different fungal growth stages, confirming a role for these phytoalexins in BTH- and BABA-induced resistance against U. pisi hampering pre- and postpenetration fungal stages.

Metabolism of stilbene phytoalexins in grapevines: Oxidation of resveratrol in single-cell cultures

Abstract: Single-cell cultures of the fungus resistant grapevine varieties Pollux and Sirius and the susceptible ones Riesling and Optima have been established to study the metabolism of the stilbene phytoalexins resveratrol and viniferin. Cells of different varieties showed morphological differences (form and mechanical resistance) and their susceptibility to treatment with debris of killed mycelium of Bot1ytis cinerea reflected the varietal resistance under natural conditions. Most cells of Riesling and Optima were killed by toxic components of the mycelium within 60 h, whereas cultures of Pollux and Sirius showed only slight damage. Two types of peroxidases have been characterized which can take resveratrol as a substrate converting it to a brown pigment. Viniferin was observed as an intermediate product. Peroxidase activity in suscpetible varieties was reduced as compared to the resistant ones. The oxidation products of resveratrol inhibited the activity of peroxidase. The function of stilbene phytoalexins as precursors of lignin-like substances is discussed.

Oil body-mediated defense against fungi: From tissues to ecology.

Abstract: Oil bodies are localized in the seed cells and leaf cells of many land plants. They have a passive function as storage organelles for lipids. We recently reported that the leaf oil body has an active function as a subcellular factory that produces an antifungal oxylipin during fungal infection in Arabidopsis thaliana. Here, we propose a model for oil body-mediated plant defense. Remarkably, senescent leaves develop oil bodies and accumulate α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3) on the oil-body membrane, which catalyze the conversion of α-linolenic acid to the phytoalexin 2-hydroxy-octadecatrienoic acid (2-HOT). The model proposes that senescent leaves actively produce antifungal oxylipins and phytoalexins, and abscised leaves contain a mixture of antifungal compounds. In natural settings, the abscised leaves with antifungal compounds accumulate in leaf litter and function to protect healthy tissues and young plants from fungal infection. Plants might have evolved this ecological function for dead leaves.

Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice.

Abstract: Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.

Differential Induction of 3-deoxyanthocyanidin Phytoalexins in Relation to Colletotrichum falcatum Resistance in Sugarcane

Abstract: Sugarcane secrets a reddish pigment presumed to contain phytoalexin compounds in the stalk tissues in response to infection of Colletotrichum falcatum, causing red rot. To identify the phytoalexin compounds involved in red rot resistance, detailed HPLC analyses were conducted using the phytoalexin standards in a set of sugarcane cultivars varying in red rot resistance, after pathogen inoculation. We found nine different 3-deoxyanthocyanidin phytoalexin compounds, which are differentially induced upon C. falcatum infection in sugarcane varieties with varying degrees of disease resistance. Among the nine compounds, four compounds were identified as luteolinidin, 5-methoxy luteolinidin, apigeninidin and arabinosyl-5-O-apigeninidin. Multifold induction of luteolinidin, apigeninidin and arabinosyl-5-O-apigeninidinin were recorded in the stalk tissues in disease resistant cvs Co 93009, BO 91 and Baragua, respectively after pathogen inoculation as compared to the susceptible cv CoC 671. In addition to the known compounds one to three unknown compounds were detected in the resistant cultivars. The susceptible cultivars exhibited a placid induction of phytoalexin compounds after pathogen infections. The results very clearly demonstrated a specific induction of 3-deoxyanthocyanidins in resistant cultivars at a higher level as compared to susceptible cultivars after C. falcatum infection in sugarcane. Total extracts of the induced phytoalexins in the calorimetric assays, also revealed similar results. Probably induction of either luteolinidin or apigeninidin compounds alone or in combination at higher concentrations in the resistant cultivars may enable the effective arrest of pathogen invasion and further development inside the stalk tissues.

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea.

Abstract: Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2'-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography-mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.

“Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

Abstract: The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.

A bestatin primes grapevine cells for augmented elicitation of the hypersensitive-like cell death and associated defense responses by methyl jasmonate

Abstract: Localized treatment of grapevine ( Vitis vinifera L. cv. Limberger) leaves with bestatin, an inhibitor of some aminopeptidases in plants and animals, augmented the sensitivity for methyl jasmonate-induced hypersensitive-like response. Enhanced resveratrol accumulation was associated with potentiated activation of genes encoding phenylalanine ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with bestatin, indicating time-dependent printing of the cells. Exogenously supplied bestatin also potentiated other characteristic elicitor-induced short- and long-term defense responses in cell suspensions of grapevine including strong medium alkalinization and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Bestatin therefore appears to be exerting its effects close to the level of transcriptional control of defense-related genes, where it might inhibit a regulatory protease. Strikingly, the ability of bestatin to potentiate grapevine PAL gene elicitation and resveratrol accumulation, emphasizes an important role for defense response potentiation in acquired plant disease resistance.

The Effect of Resveratrol on Swarming Differentiation and the Expression of Some Virulence Factors in Proteus vulgaris

Abstract: Resveratrol (3,5,4'-trihydroxy- trans -stilbene) is a stilbenoid, a type of natural phenol, and a phytoalexin with anti-inflammatory and antioxidant activities. It is produced naturally by several plants especially the roots of the Japanese Knotweed when under attack by pathogens such as bacteria or fungi. In this study we have verified that resveratrol has activity against Proteus vulgaris , an important pathogen infecting the urinary tract by investigating its effect on swarming and some virulence factor expression(haemolysin and urease).Swarming inhibition was determined on Luria Bertani agar with and without resveratrol and then bacteria was harvested to assay cell length and the production of haemolysin and urease. Resveratrol significantly inhibited swarming and virulence factor expression but its effect on growth rate was not significant. Keywords: Resveratrol, Proteus vulgaris , phytoalexin, haemolysin, urease.