The putative H3K36 demethylase BcKDM1 affects virulence, stress responses and photomorphogenesis in Botrytis cinerea.

The Leotiomycete Botrytis cinerea is a high-impact plant pathogen causing gray mold disease in a wide range of dicotyledonous species. Besides its efficient strategies to cause disease - either by being highly aggressive leading to rapid destruction of plant tissues or by keeping hidden for certain periods before damaging the host - the fungus is well-adapted to the changing environmental conditions due to different modes of reproduction for dispersal (macroconidia), survival (sclerotia) or adaptation (ascospores formed in the apothecia). The screening of a collection of B. cinerea mutants generated by Agrobacterium tumefaciens-mediated transformation (ATMT) has revealed a number of virulence-attenuated mutants. In the avirulent mutant PA2810 the inserted T-DNA disrupts the gene encoding a putative histone 3 lysine 36 (H3K36)-specific demethylase (BcKDM1). Targeted mutagenesis of bckdm1 confirmed the gene-phenotype linkage and indicated that BcKDM1, despite its role in virulence (critical for penetration), is required for coping with excessive light, oxidative stress and for proper expression of light-responsive genes and photomorphogenesis. Thus, bckdm1 loss-of-function mutants produce sclerotia under unfavorable conditions such as in the light. Notably, mutants expressing a truncated BcKDM1 (bckdm1991aa) showed deviating phenotypes from deletion (Δbckdm1) and demethylase-deficient (bckdm1H360A) mutants but also from the wild type, thereby indicating the importance of the C-terminal region for some developmental processes. This effect may be specific to B. cinerea as the orthologs from other Ascomycetes cannot replace BcKDM1.

Manipulation of cytokinin level in the ergot fungus Claviceps purpurea emphasizes its contribution to virulence.

Pathogen-derived cytokinins (CKs) have been recognized as important virulence factor in several host-pathogen interactions and it was demonstrated multiple times that phytopathogenic fungi form CKs via the tRNA degradation pathway. In contrast to previous studies, the focus of this study is on the second step of CK formation and CK degradation to improve our understanding of the biosynthesis in fungi on the one hand, and to understand CK contribution to the infection process of Claviceps purpurea on the other hand. The ergot fungus Claviceps purpurea is a biotrophic phytopathogen with a broad host range including economically important crops causing harvest intoxication upon infection. Its infection process is restricted to unfertilized ovaries without causing macroscopic defense symptoms. Thus, sophisticated host manipulation strategies are implicated. The cytokinin (CK) plant hormones are known to regulate diverse plant cell processes, and several plant pathogens alter CK levels during infection. C. purpurea synthesizes CKs via two mechanisms, and fungus-derived CKs influence the host-pathogen interaction but not fungus itself. CK deficiency in fungi with impact on virulence has only been achieved to date by deletion of a tRNA-ipt gene that is also involved in a process of translation regulation. To obtain a better understanding of CK biosynthesis and CKs' contribution to the plant-fungus interaction, we applied multiple approaches to generate strains with altered or depleted CK content. The first approach is based on deletion of the two CK phosphoribohydrolase (LOG)-encoding genes, which are believed to be essential for the release of active CKs. Single and double deletion strains were able to produce all types of CKs. Apparently, log gene products are dispensable for the formation of CKs and so alternative activation pathways must be present. The CK biosynthesis pathway remains unaffected in the second approach, because it is based on heterologous overexpression of CK-degrading enzymes from maize (ZmCKX1). Zmckx1 overexpressing C. purpurea strains shows strong CKX activity and drastically reduced CK levels. The strains are impaired in virulence, which reinforces the assumption that fungal-derived CKs are crucial for full virulence. Taken together, this study comprises the first analysis of a log depletion mutant that proved the presence of alternative cytokinin activation pathways in fungi and showed that heterologous CKX expression is a suitable approach for CK level reduction.

Brachypodium distachyon as alternative model host system for the ergot fungus Claviceps purpurea.

To investigate its susceptibility to ergot infection, we inoculated Brachypodium distachyon with Claviceps purpurea and compared the infection symptoms with those on rye (Secale cereale). We showed that, after inoculation of Brachypodium with Claviceps, the same disease symptoms occurred in comparable temporal and spatial patterns to those on rye. The infection rate of Claviceps on this host was reduced compared with rye, but the disease could be surveyed by fungal genomic DNA quantification. Mutants of Claviceps which were virulence attenuated on rye were also affected on Brachypodium. We were able to show that pathogenesis-related gene expression changed in a typical manner for biotrophic pathogen attack. Our results indicated that the Claviceps-Brachypodium interaction was dependent on salicylic acid, cytokinin and auxin. We consider Brachypodium to be a suitable and useful alternative host; the increased sensitivity compared with rye will be valuable for the identification of infection mechanisms. Future progess in understanding the Claviceps-plant interaction will be facilitated by the use of a well-characterized model host system.

The Protein Disulfide Isomerase of Botrytis cinerea: An ER Protein Involved in Protein Folding and Redox Homeostasis Influences NADPH Oxidase Signaling Processes.

Botrytis cinerea is a filamentous plant pathogen, which infects hundreds of plant species; within its lifestyle, the production of reactive oxygen species (ROS) and a balanced redox homeostasis are essential parameters. The pathogen is capable of coping with the plant's oxidative burst and even produces its own ROS to enhance the plant's oxidative burst. Highly conserved NADPH oxidase (Nox) complexes produce the reactive molecules. The membrane-associated complexes regulate a large variety of vegetative and pathogenic processes. Besides their commonly accepted function at the plasma membrane, recent studies reveal that Nox complexes are also active at the membrane of the endoplasmic reticulum. In this study, we identified the essential ER protein BcPdi1 as new interaction partner of the NoxA complex in B. cinerea. Mutants that lack this ER chaperone display overlapping phenotypes to mutants of the NoxA signaling pathway. The protein appears to be involved in all major developmental processes, such as the formation of sclerotia, conidial anastomosis tubes and infection cushions (IC's) and is needed for full virulence. Moreover, expression analyses and reporter gene studies indicate that BcPdi1 affects the redox homeostasis and unfolded protein response (UPR)-related genes. Besides the close association between BcPdi1 and BcNoxA, interaction studies provide evidence that the ER protein might likewise be involved in Ca2+ regulated processes. Finally, we were able to show that the potential key functions of the protein BcPdi1 might be affected by its phosphorylation state.

Cross-talk of the biotrophic pathogen Claviceps purpurea and its host Secale cereale.

BACKGROUND: The economically important Ergot fungus Claviceps purpurea is an interesting biotrophic model system because of its strict organ specificity (grass ovaries) and the lack of any detectable plant defense reactions. Though several virulence factors were identified, the exact infection mechanisms are unknown, e.g. how the fungus masks its attack and if the host detects the infection at all. RESULTS: We present a first dual transcriptome analysis using an RNA-Seq approach. We studied both, fungal and plant gene expression in young ovaries infected by the wild-type and two virulence-attenuated mutants. We can show that the plant recognizes the fungus, since defense related genes are upregulated, especially several phytohormone genes. We present a survey of in planta expressed fungal genes, among them several confirmed virulence genes. Interestingly, the set of most highly expressed genes includes a high proportion of genes encoding putative effectors, small secreted proteins which might be involved in masking the fungal attack or interfering with host defense reactions. As known from several other phytopathogens, the C. purpurea genome contains more than 400 of such genes, many of them clustered and probably highly redundant. Since the lack of effective defense reactions in spite of recognition of the fungus could very well be achieved by effectors, we started a functional analysis of some of the most highly expressed candidates. However, the redundancy of the system made the identification of a drastic effect of a single gene most unlikely. We can show that at least one candidate accumulates in the plant apoplast. Deletion of some candidates led to a reduced virulence of C. purpurea on rye, indicating a role of the respective proteins during the infection process. CONCLUSIONS: We show for the first time that- despite the absence of effective plant defense reactions- the biotrophic pathogen C. purpurea is detected by its host. This points to a role of effectors in modulation of the effective plant response. Indeed, several putative effector genes are among the highest expressed genes in planta.

Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence.

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development.

Localization of ergot alkaloids in sclerotia of Claviceps purpurea by matrix-assisted laser desorption/ionization mass spectrometry imaging.

The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 µm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction. Graphical abstract Workflow for identification and spatial localization of ergot alkaloids in infected rye grains.

The Epipolythiodiketopiperazine Gene Cluster in Claviceps purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines.

Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP) toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster.

BcIqg1, a fungal IQGAP homolog, interacts with NADPH oxidase, MAP kinase and calcium signaling proteins and regulates virulence and development in Botrytis cinerea.

NADPH oxidases (Nox) produce reactive oxygen species (ROS) in multicellular eukaryotic organisms. They trigger defense reactions ('oxidative burst') - in phagocytes and plant cells -, and are involved in a broad range of differentiation processes. Fungal Nox-complexes play a central role in vegetative, sexual and pathogenic processes. In contrast to mammalian systems, knowledge is limited about composition, localisation and connection to major signaling cascades in fungi. Here, we characterize a fungal homolog of the RasGAP scaffold protein IQGAP, which links several major signaling processes, including Nox in mammalian cell lines. We show that BcIqg1 interacts directly with a cytosolic, regulatory component (BcRac) and a membrane-associated subunit (BcNoxD) of a Nox-complex in the pathogen Botrytis cinerea. Thus, this protein may be a scaffold that mediates interaction of the catalytic subunits with the regulator BcNoxR. The protein interacts with modules of the MAP kinase- and calcium-dependent signaling pathways. Functional analysis of BcIqg1 substantiated its involvement in different signaling pathways. It mediates the Ca(2+) -triggered nuclear translocation of - BcCRZ1 and the MAP kinase BcBmp1. BcIqg1 is involved in resistance against oxidative and membrane stress and is required for several developmental processes including formation of sclerotia, conidial anastomosis tubes and infection cushions as well as for virulence.

Reactive oxygen species in development and infection processes.

Reactive oxygen species (ROS) are important signaling molecules that affect vegetative and pathogenic processes in pathogenic fungi. There is growing evidence that ROS are not only secreted during the interaction of host and pathogen but also involved in tightly controlled intracellular processes. The major ROS producing enzymes are NADPH oxidases (Nox). Recent investigations in fungi revealed that Nox-activity is responsible for the formation of infection structures, cytoskeleton architecture as well as interhyphal communication. However, information about the localization and site of action of the Nox complexes in fungi is limited and signaling pathways and intracellular processes affected by ROS have not been fully elucidated. This review focuses on the role of ROS as signaling molecules in fungal "model" organisms: it examines the role of ROS in vegetative and pathogenic processes and gives special attention to Nox complexes and their function as important signaling hubs.

Functional characterization of the first filamentous fungal tRNA-isopentenyltransferase and its role in the virulence of Claviceps purpurea.

In plants, cytokinins (CKs) are synthesized de novo or by the degradation of modified tRNAs. Recently, the first fungal de novo pathway was identified within the plant pathogen Claviceps purpurea. As the deletion of the de novo pathway did not lead to a complete loss of CKs, this work focuses on the tRNA-modifying protein tRNA-isopentenyltransferase (CptRNA-IPT). The contribution of this enzyme to the CK pool of Claviceps and the role of CKs in the host-pathogen interaction are emphasized. The effects of the deletion of cptRNA-ipt and the double deletion of cptRNA-ipt and the key gene of de novo biosynthesis cpipt-log on growth, CK biosynthesis and virulence were analyzed. In addition, the sites of action of CptRNA-IPT were visualized using reporter gene fusions. In addition to CK-independent functions, CptRNA-IPT was essential for the biosynthesis of cis-zeatin (cZ) and contributed to the formation of isopentenyladenine (iP) and trans-zeatin (tZ). Although ΔcptRNA-ipt was reduced in virulence, the 'CK-free' double deletion mutant was nearly apathogenic. The results prove a redundancy of the CK biosynthesis pathway in C. purpurea for iP and tZ formation. Moreover, we show, for the first time, that CKs are required for the successful establishment of a host-fungus interaction.

Chasing stress signals - Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea.

Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA.

Identification and characterization of the ergochrome gene cluster in the plant pathogenic fungus Claviceps purpurea.

BACKGROUND: Claviceps purpurea is a phytopathogenic fungus infecting a broad range of grasses including economically important cereal crop plants. The infection cycle ends with the formation of the typical purple-black pigmented sclerotia containing the toxic ergot alkaloids. Besides these ergot alkaloids little is known about the secondary metabolism of the fungus. Red anthraquinone derivatives and yellow xanthone dimers (ergochromes) have been isolated from sclerotia and described as ergot pigments, but the corresponding gene cluster has remained unknown. Fungal pigments gain increasing interest for example as environmentally friendly alternatives to existing dyes. Furthermore, several pigments show biological activities and may have some pharmaceutical value. RESULTS: This study identified the gene cluster responsible for the synthesis of the ergot pigments. Overexpression of the cluster-specific transcription factor led to activation of the gene cluster and to the production of several known ergot pigments. Knock out of the cluster key enzyme, a nonreducing polyketide synthase, clearly showed that this cluster is responsible for the production of red anthraquinones as well as yellow ergochromes. Furthermore, a tentative biosynthetic pathway for the ergot pigments is proposed. By changing the culture conditions, pigment production was activated in axenic culture so that high concentration of phosphate and low concentration of sucrose induced pigment syntheses. CONCLUSIONS: This is the first functional analysis of a secondary metabolite gene cluster in the ergot fungus besides that for the classical ergot alkaloids. We demonstrated that this gene cluster is responsible for the typical purple-black color of the ergot sclerotia and showed that the red and yellow ergot pigments are products of the same biosynthetic pathway. Activation of the gene cluster in axenic culture opened up new possibilities for biotechnological applications like the dye production or the development of new pharmaceuticals.

Update on Nox function, site of action and regulation in Botrytis cinerea.

BACKGROUND: The production of reactive oxygen species (ROS) and a balanced redox homeostasis are essential parameters, which control the infection process of the plant pathogen Botrytis cinerea. The necrotrophic fungus is able to cope with the plants' oxidative burst and even produces its own ROS to overcome the plants' defense barrier. Major enzyme complexes, which are responsible for the production of superoxide, are NADPH oxidase (Nox) complexes. They play a central role in various growth, differentiation and pathogenic processes. However, information about their regulation and the integration in the complex signaling network of filamentous fungi is still scarce. RESULTS: In this work, we give an update on Nox structure, function, site of action and regulation. We show that functionality of the catalytic Nox-subunits seems to be independent from their transcriptional regulation and that the membrane orientation of BcNoxA would allow electron transport inside the ER. Following previous studies, which provided evidence for distinct functions of the NoxA complex inside the ER, we highlight in this work that the N-terminus of BcNoxA is essential for these functions. Finally, we elucidate the role of BcNoxD and BcNoxB inside the ER by complementing the deletion mutants with ER bound alleles. CONCLUSIONS: This study provides a deeper analysis of the Nox complexes in B. cinerea. Besides new insights in the overall regulation of the complexes, we provide further evidence that the NoxA complex has a predominant role inside the ER, while the NoxB complex is mainly important outside the ER, likely at the plasma membrane. By considering all other putative Nox complex members, we propose a putative model, which describes the distinct complex pattern upon certain differentiation processes.

Unraveling the Function of the Response Regulator BcSkn7 in the Stress Signaling Network of Botrytis cinerea.

Important for the lifestyle and survival of every organism is the ability to respond to changing environmental conditions. The necrotrophic plant pathogen Botrytis cinerea triggers an oxidative burst in the course of plant infection and therefore needs efficient signal transduction to cope with this stress. The factors involved in this process and their precise roles are still not well known. Here, we show that the transcription factor Bap1 and the response regulator (RR) B. cinerea Skn7 (BcSkn7) are two key players in the oxidative stress response (OSR) of B. cinerea; both have a major influence on the regulation of classical OSR genes. A yeast-one-hybrid (Y1H) approach proved direct binding to the promoters of gsh1 and grx1 by Bap1 and of glr1 by BcSkn7. While the function of Bap1 is restricted to the regulation of oxidative stress, analyses of Δbcskn7 mutants revealed functions beyond the OSR. Involvement of BcSkn7 in development and virulence could be demonstrated, indicated by reduced vegetative growth, impaired formation of reproductive structures, and reduced infection cushion-mediated penetration of the host by the mutants. Furthermore, Δbcskn7 mutants were highly sensitive to oxidative, osmotic, and cell wall stress. Analyses of Δbap1 bcskn7 double mutants indicated that loss of BcSkn7 uncovers an underlying phenotype of Bap1. In contrast to Saccharomyces cerevisiae, the ortholog of the glutathione peroxidase Gpx3p is not required for nuclear translocation of Bap1. The presented results contribute to the understanding of the OSR in B. cinerea and prove that it differs substantially from that of yeast, demonstrating the complexity and versatility of components involved in signaling pathways.

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea.

Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot-infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT-LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans-zeatin, a reaction catalysed by a CK-specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt-log. Deletion of cpipt-log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper-sporulating phenotype, implying that CKs are environmental factors influencing fungal development.

BcNoxD, a putative ER protein, is a new component of the NADPH oxidase complex in Botrytis cinerea.

NADPH oxidases (Nox) are major enzymatic producer of reactive oxygen species (ROS). In fungi these multi-enzyme complexes are involved in sexual differentiation and pathogenicity. However, in contrast to mammalian systems, the composition and recruitment of the fungal Nox complexes are unresolved. Here we introduce a new Nox component, the membrane protein NoxD in the grey mold fungus Botrytis cinerea. It has high homology to the ER protein Pro41 from Sordaria macrospora, similar functions to the catalytic Nox subunit BcNoxA in differentiation and pathogenicity, and shows similarities to phagocytic p22phox. BcNoxA and BcNoxD interact with each other. Both proteins are involved in pathogenicity, fusion of conidial anastomosis tubes (CAT) and formation of sclerotia and conidia. These data support our earlier view based on localization studies, for an ER-related function of the Nox complex. We present the first evidence that some functions of the BcNoxA complex are indeed linked to the ER, while others clearly require export from the ER.

Biosynthetic pathways of ergot alkaloids.

Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes.

A new and reliable method for live imaging and quantification of reactive oxygen species in Botrytis cinerea: technological advancement.

Reactive oxygen species (ROS) are produced in conserved cellular processes either as by-products of the cellular respiration in mitochondria, or purposefully for defense mechanisms, signaling cascades or cell homeostasis. ROS have two diametrically opposed attributes due to their highly damaging potential for DNA, lipids and other molecules and due to their indispensability for signaling and developmental processes. In filamentous fungi, the role of ROS in growth and development has been studied in detail, but these analyses were often hampered by the lack of reliable and specific techniques to monitor different activities of ROS in living cells. Here, we present a new method for live cell imaging of ROS in filamentous fungi. We demonstrate that by use of a mixture of two fluorescent dyes it is possible to monitor H2O2 and superoxide specifically and simultaneously in distinct cellular structures during various hyphal differentiation processes. In addition, the method allows for reliable fluorometric quantification of ROS. We demonstrate that this can be used to characterize different mutants with respect to their ROS production/scavenging potential.

Redox systems in Botrytis cinerea: impact on development and virulence.

The thioredoxin system is of great importance for maintenance of cellular redox homeostasis. Here, we show that it has a severe influence on virulence of Botrytis cinerea, demonstrating that redox processes are important for host-pathogen interactions in this necrotrophic plant pathogen. The thioredoxin system is composed of two enzymes, the thioredoxin and the thioredoxin reductase. We identified two genes encoding for thioredoxins (bctrx1, bctrx2) and one gene encoding for a thioredoxin reductase (bctrr1) in the genome of B. cinerea. Knockout mutants of bctrx1 and bctrr1 were severely impaired in virulence and more sensitive to oxidative stress. Additionally, Δbctrr1 showed enhanced H2O2 production and retarded growth. To investigate the impact of the second major cellular redox system, glutathione, we generated deletion mutants for two glutathione reductase genes. The effects were only marginal; deletion of bcglr1 resulted in reduced germination and, correspondingly, to retarded infection as well as reduced growth on minimal medium, whereas bcglr2 deletion had no distinctive phenotype. In summary, we showed that the balanced redox status maintained by the thioredoxin system is essential for development and pathogenesis of B. cinerea, whereas the second major cellular redox system, the glutathione system, seems to have only minor impact on these processes.