Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-α-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-α-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.

The sesquiterpene botrydial from Botrytis cinerea induces phosphatidic acid production in tomato cell suspensions.

MAIN CONCLUSION: The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6.

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle.

Natural variation in the VELVET gene bcvel1 affects virulence and light-dependent differentiation in Botrytis cinerea.

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the full-length protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Δbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Δbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Δbcvel1 mutants to nutrient starvation conditions.

Biotransformation of clovane derivatives. Whole cell fungi mediated domino synthesis of rumphellclovane A.

Here we describe the biotransformation of clovane derivatives by filamentary fungi Pestalotiopsis palustris and Penicillium minioluteum, and the application of the latter to the synthesis and determination of the absolute configuration of rumphellclovane A (2). Methoxyclovanol (1), a growth inhibitor of the phytopathogen Botrytis cinerea, is metabolised by P. palustris to yield rumphellclovane A (2), a natural compound recently isolated from the gorgonian coral Rumphella antipathies, two new compounds, (1R,2S,5S,8R,9S,10R)-2-methoxyclovane-9,10-diol (5) and (1S,2S,5S,7R,8R,9R)-2-methoxyclovane-7,9-diol (6), hydroxylated in positions not easily accessed by classic synthetic chemistry, and clovanodiols 3 and 4. P. minioluteum is able to selectively transform methoxyclovanol (1) into clovanodiols 3 and 4 and, in turn, lactone 8, the putative intermediate in the above mentioned synthesis of rumphellclovane A (2), into compound 2 via a domino process. The ability of P. minioluteum to carry out the cleavage of ethers on clovane derivatives is also evaluated.

The mitogen-activated protein kinase BcSak1 of Botrytis cinerea is required for pathogenic development and has broad regulatory functions beyond stress response.

The mitogen-activated protein kinase (MAPK) BcSak1 of Botrytis cinerea is activated upon exposure to H(2)O(2) and, hence, might be involved in coping with oxidative stress during infection. However, beside osmotic and oxidative stress sensitivity, Δbcsak1 mutants have a pleiotropic phenotype, as they do not produce conidia and are unable to penetrate unwounded host tissue. In this study, the role of BcSak1 was investigated in the stress response and during infection of French beans by Botrytis cinerea. Using a macroarray approach, it was shown that BcSak1 is only marginally involved in the specific oxidative stress response. In fact, the induction of several genes after oxidative stress treatment is BcSak1-dependent, but most of these genes are also induced under conditions of osmotic stress. The majority of genes regulated by BcSak1 are not involved in the stress response at all. Using a translational fusion of BcSak1 to green fluorescent protein, it was shown clearly that the localization of this MAPK depends on the type of stress being applied; it associates rapidly to the nucleus only under osmotic stress. Therefore, a model is proposed in which BcSak1 acts in the cytosol by activation of one or more transcription factors under oxidative stress and, at the same time, it reacts to osmotic stress by migrating to the nucleus. Interestingly, the MAPK is also involved in the regulation of secondary metabolism, as the major phytotoxins secreted by this fungus are reduced in the Δbcsak1 deletion mutant. Experiments done in planta underlined the essential role of BcSak1 in the early stages of infection, when it translocates to the nucleus and then changes to cytosolic distribution during hyphal growth within the tissue.

BcAtf1, a global regulator, controls various differentiation processes and phytotoxin production in Botrytis cinerea.

Atf1-homologous basic region leucine zipper (bZIP) transcription factors are known to act downstream of the stress-activated mitogen-activated protein kinase (SAPK) cascade in mammals, as well as in several fungi; they regulate the transcription of genes involved in the general stress response. Functional analyses of BcAtf1 in Botrytis cinerea show that it is also connected to the SAPK BcSak1, as it shares several stress response target genes. However, Δbcatf1 mutants are not hypersensitive to osmotic or oxidative stress, as are Δbcsak1 mutants. Both BcSak1 and BcAtf1 are regulators of differentiation, but their roles in these processes are almost inverse as, in contrast with Δbcsak1, Δbcatf1 mutants are significantly impaired in conidia production and do not differentiate any sclerotia. They show extremely vigorous growth in axenic culture, with a thick layer of aerial hyphae and a marked increase in colonization efficiency on different host plants and tissues. In addition, the sensitivity to cell wall-interfering agents is increased strongly. Microarray analyses demonstrate that the loss of BcAtf1 leads to extensive transcriptional changes: apart from stress response genes, the expression of a broad set of genes, probably involved in primary metabolism, cell wall synthesis and development, is affected by BcAtf1. Unexpectedly, BcAtf1 also controls secondary metabolism: the mutant contains significantly elevated levels of phytotoxins. These data indicate that BcAtf1 controls a diversity of cellular processes and has broad regulatory functions.

The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial.

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.

The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response on plant tissues and its action is modulated by salicylic acid and jasmonic acid signaling.

Botrytis cinerea, as a necrotrophic fungus, kills host tissues and feeds on the remains. This fungus is able to induce the hypersensitive response (HR) on its hosts, thus taking advantage on the host's defense machinery for generating necrotic tissues. However, the identity of HR effectors produced by B. cinerea is not clear. The aim of this work was to determine whether botrydial, a phytotoxic sesquiterpene produced by B. cinerea, is able to induce the HR on plant hosts, using Arabidopsis thaliana as a model. Botrydial induced the expression of the HR marker HSR3, callose deposition, and the accumulation of reactive oxygen species and phenolic compounds. Botrydial also induced the expression of PR1 and PDF1.2, two pathogenesis-related proteins involved in defense responses regulated by salicylic acid (SA) and jasmonic acid (JA), respectively. A. thaliana and tobacco plants defective in SA signaling were more resistant to botrydial than wild-type plants, as opposed to A. thaliana plants defective in JA signaling, which were more sensitive. It can be concluded that botrydial induces the HR on its hosts and its effects are modulated by host signaling pathways mediated by SA and JA.

Ultra-fast gradient LC method for omeprazole analysis using a monolithic column: assay development, validation, and application to the quality control of omeprazole enteric-coated pellets.

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.

Fungal terpene metabolites: biosynthetic relationships and the control of the phytopathogenic fungus Botrytis cinerea.

The structures and biosynthesis of the sesquiterpenoid metabolites of Botrytis cinerea and their relationship to the presilphiperfolanes are reviewed. The development of a novel strategy for the control of this phytopathogenic fungus based on analogues of these metabolites is described. There are 75 references.

Metabolites from Eutypa species that are pathogens on grapes.

Structural and synthetic studies of the metabolites isolated from Eutypa lata are reviewed. This fungus is the causative agent of Eutypa dieback disease, also known as eutyposis or dying-arm disease, a perennial canker that affects grapevines and many other woody fruit plants. The review, which encompasses all the literature in this field up to the present and in which 76 references are cited, also includes a detailed study of the biological activity of the metabolites, especially the role of toxins in the development of the plant disease. Some aspects of the synthesis and biosynthesis of these metabolites and related compounds are discussed.