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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 (bckdm1 991aa ) showed deviating phenotypes from deletion (Δbckdm1) and demethylase-deficient (bckdm1 H360A ) 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Botrytis cinerea VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their molecular basis. Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed. VELVET mutants showed reduced release of citric acid, the major acid secreted by the wild-type, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with 15 N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation. The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Small-molecule signaling is one major mode of communication within the polymicrobial consortium of soil and rhizosphere. While microbial secondary metabolite (SM) production and responses of individual species have been studied extensively, little is known about potentially conserved roles of SM signals in multilayered symbiotic or antagonistic relationships. Here, we characterize the SM-mediated interaction between the plant-pathogenic bacterium Ralstonia solanacearum and the two plant-pathogenic fungi Fusarium fujikuroi and Botrytis cinerea We show that cellular differentiation and SM biosynthesis in F. fujikuroi are induced by the bacterially produced lipopeptide ralsolamycin (synonym ralstonin A). In particular, fungal bikaverin production is induced and preferentially accumulates in fungal survival spores (chlamydospores) only when exposed to supernatants of ralsolamycin-producing strains of R. solanacearum Although inactivation of bikaverin biosynthesis moderately increases chlamydospore invasion by R. solanacearum , we show that other metabolites such as beauvericin are also induced by ralsolamycin and contribute to suppression of R. solanacearum growth in vitro Based on our findings that bikaverin antagonizes R. solanacearum and that ralsolamycin induces bikaverin biosynthesis in F. fujikuroi , we asked whether other bikaverin-producing fungi show similar responses to ralsolamycin. Examining a strain of B. cinerea that horizontally acquired the bikaverin gene cluster from Fusarium , we found that ralsolamycin induced bikaverin biosynthesis in this fungus. Our results suggest that conservation of microbial SM responses across distantly related fungi may arise from horizontal transfer of protective gene clusters that are activated by conserved regulatory cues, e.g., a bacterial lipopeptide, providing consistent fitness advantages in dynamic polymicrobial networks. IMPORTANCE Bacteria and fungi are ubiquitous neighbors in many environments, including the rhizosphere. Many of these organisms are notorious as economically devastating plant pathogens, but little is known about how they communicate chemically with each other. Here, we uncover a conserved antagonistic communication between the widespread bacterial wilt pathogen Ralstonia solanacearum and plant-pathogenic fungi from disparate genera, Fusarium and Botrytis Exposure of Fusarium fujikuroi to the bacterial lipopeptide ralsolamycin resulted in production of the antibacterial metabolite bikaverin specifically in fungal tissues invaded by Ralstonia Remarkably, ralsolamycin induction of bikaverin was conserved in a Botrytis cinerea isolate carrying a horizontally transferred bikaverin gene cluster. These results indicate that horizontally transferred gene clusters may carry regulatory prompts that contribute to conserved fitness functions in polymicrobial environments., (Copyright © 2018 Spraker et al.)

Botrytis cinerea is a plant pathogenic fungus with a broad host range. Due to its rapid growth and reproduction by asexual spores (conidia), which increases the inoculum pressure, the fungus is a serious problem in different fields of agriculture. The formation of the conidia is promoted by light, whereas the formation of sclerotia as survival structures occurs in its absence. Based on this observation, putative transcription factors (TFs) whose expression is induced upon light exposure have been considered as candidates for activating conidiation and/or repressing sclerotial development. Previous studies reported on the identification of six light-responsive TFs (LTFs), and two of them have been confirmed as crucial developmental regulators: BcLTF2 is the positive regulator of conidiation, whose expression is negatively regulated by BcLTF1. Here, the functional characterization of the four remaining LTFs is reported. BcLTF3 has a dual function, as it represses conidiophore development by repressing bcltf2 in light and darkness, and is moreover essential for conidiogenesis. In bcltf3 deletion mutants conidium initials grow out to hyphae, which develop secondary conidiophores. In contrast, no obvious functions could be assigned to BcLTF4, BcLTF5 and BcLTF6 in these experiments. BcREG1, previously reported to be required for virulence and conidiogenesis, has been re-identified as light-responsive transcriptional regulator. Studies with bcreg1 overexpression strains indicated that BcREG1 differentially affects conidiation by acting as a repressor of BcLTF2-induced conidiation in the light and as an activator of a BcLTF2-independent conidiation program in the dark.

Fungi, like other organisms, actively sense the environmental light conditions in order to drive adaptive responses, including protective mechanisms against the light-associated stresses, and to regulate development. Ecological niches are characterized by different light regimes, for instance light is absent underground, and light spectra from the sunlight are changed underwater or under the canopy of foliage due to the absorption of distinct wavelengths by bacterial, algal and plant pigments. Considering the fact that fungi have evolved to adapt to their habitats, the complexities of their 'visual' systems may vary significantly. Fungi that are pathogenic on plants experience a special light regime because the host always seeks the optimum light conditions for photosynthesis - and the pathogen has to cope with this environment. When the pathogen lives under the canopy and is indirectly exposed to sunlight, it is confronted with an altered light spectrum enriched for green and far-red light. Botrytis cinerea, the gray mold fungus, is an aggressive plant pathogen mainly infecting the above-ground parts of the plant. As outlined in this review, the Leotiomycete maintains a highly sophisticated light signaling machinery, integrating (near)-UV, blue, green, red and far-red light signals by use of at least eleven potential photoreceptors to trigger a variety of responses, i.e. protection (pigmentation, enzymatic systems), morphogenesis (conidiation, apothecial development), entrainment of a circadian clock, and positive and negative tropism of multicellular (conidiophores, apothecia) and unicellular structures (conidial germ tubes). In that sense, 'looking through the eyes' of this plant pathogen will expand our knowledge of fungal photobiology., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system. IMPORTANCE Botrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation)., (Copyright © 2017 American Society for Microbiology.)

The gray mold fungus Botrytis cinerea features a wide host range and causes severe economic losses, making it an important object for molecular research. Thus far, genetic modification of the fungus mainly is relied on two selection systems (nourseothricin and hygromycin), while other selection systems hold significant disadvantages. To broaden the spectrum of available molecular tools, a new selection system based on the cheap and widely used fungicide fenhexamid (hydroxyanilide group) was established. Fenhexamid specifically targets the 3-ketoreductase ERG27 from the ergosterol biosynthesis pathway. We generated a set of expression vectors suitable for deletion or expression of genes of interest (GOIs) in B. cinerea based on fenhexamid-insensitive ERG27 variants. Expression of BcERG27 F412I and Fusarium fujikuroi ERG27 in the sensitive B. cinerea strain B05.10 causes resistance towards fenhexamid (fenR) and allows for the selection of transformants and their genetic purification. A modified split-marker approach facilitates the site-specific integration and expression of GOIs at the bcerg27 locus. No undesired secondary phenotypes regarding virulence, stress responses, the formation of reproductive structures or conidial germination were observed in strains expressing fenhexamid-insensitive ERG27 variants. Thus, the fenR system represents a third reliable selection system for genetic modifications of fenhexamid-sensitive B. cinerea strains., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulate asexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. As no orthologues of known regulators of conidiation (e.g., Aspergillus nidulans BrlA, Neurospora crassa FL) exist in the Leotiomycetes, we initiated a de novo approach to identify the functional counterpart in B. cinerea. The search revealed the light-responsive C2H2 transcription factor BcLTF2 whose expression - usually restricted to light conditions - is necessary and sufficient to induce conidiation and simultaneously to suppress sclerotial development. Light-induced expression of bcltf2 is mediated via a so far unknown pathway, and is attenuated in a (blue) light-dependent fashion by the White Collar complex, BcLTF1 and the VELVET complex. Mutation of either component leads to increased bcltf2 expression and causes light-independent conidiation (always conidia phenotype). Hence, the tight regulation of bcltf2 governs the balance between vegetative growth that allows for the colonization of the substrate and subsequent reproduction via conidia in the light. The orthologue ssltf2 in the closely related species Sclerotinia sclerotiorum is not significantly expressed suggesting that its deregulation may cause the lack of the conidiation program in this fungus., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Filamentous (F-) actin is an integral part of the cytoskeleton allowing for cell growth, intracellular motility, and cytokinesis of eukaryotic cells. Its assembly from G-actin monomers and its disassembly are tightly regulated processes involving a number of actin-binding proteins (ABPs) such as F-actin nucleators and cross-linking proteins. F-actin capping protein (CP) is an alpha/beta heterodimer known from yeast and higher eukaryotes to bind to the fast growing ends of the actin filaments stabilizing them. In this study, we identified the orthologs of the two CP subunits, named BcCPA1 and BcCPB1, in the plant pathogenic fungus Botrytis cinerea and showed that the two proteins physically interact in a yeast two-hybrid approach. GFP-BcCPA1 fusion proteins were functional and localized to the assumed sites of F-actin accumulation, i.e. to the hyphal tips and the sites of actin ring formation. Deletion of bccpa1 had a profound effect on hyphal growth, morphogenesis, and virulence indicating the importance of F-actin capping for an intact actin cytoskeleton. As polarized growth - unlike septum formation - is impaired in the mutants, it can be concluded that the organization and/or localization of actin patches and cables are disturbed rather than the functionality of the actin rings., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Botrytis cinerea is the causal agent of gray mold disease in various plant species and produces grayish macroconidia and/or black sclerotia at the end of the infection cycle. It has been suggested that the pigmentation is due to the accumulation of 1,8-dihydroxynaphthalene (DHN) melanin. To unravel its basis and regulation, the putative melanogenic and regulatory genes were identified and functionally characterized. Unlike other DHN melanin-producing fungi, B. cinerea and other Leotiomycetes contain two key enzyme (PKS)-encoding enzymes. Bcpks12 and bcpks13 are developmentally regulated and are required for melanogenesis in sclerotia and conidia respectively. BcYGH1 converts the BcPKS13 product and contributes thereby to conidial melanogenesis. In contrast, enzymes acting downstream in conversion of the PKS products (BcBRN2, BcSCD1 and BcBRN1) are required for both, sclerotial and conidial melanogenesis, suggesting that DHN melanogenesis in B. cinerea follows a non-linear pathway that is rather unusual for secondary metabolic pathways. Regulation of the melanogenic genes involves three pathway-specific transcription factors (TFs) that are clustered with bcpks12 or bcpks13 and other developmental regulators such as light-responsive TFs. Melanogenic genes are dispensable in vegetative mycelia for proper growth and virulence. However, DHN melanin is considered to contribute to the longevity of the reproduction structures., (© 2015 John Wiley & Sons Ltd.)

Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light-dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia formation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulence.

In phytopathogenic fungi the establishment and maintenance of polarity is not only essential for vegetative growth and differentiation, but also for penetration and colonization of host tissues. We investigated orthologs of members of the yeast polarity complex in the grey mould fungus Botrytis cinerea: the scaffold proteins Bem1 and Far1, the GEF (guanine nucleotide exchange factor) Cdc24, and the formin Bni1 (named Sep1 in B. cinerea). BcBem1 does not play an important role in regular hyphal growth, but has significant impact on spore formation and germination, on the establishment of conidial anastomosis tubes (CATs) and on virulence. As in other fungi, BcBem1 interacts with the GEF BcCdc24 and the formin BcSep1, indicating that in B. cinerea the apical complex has a similar structure as in yeast. A functional analysis of BcCdc24 suggests that it is essential for growth, since it was not possible to obtain homokaryotic deletion mutants. Heterokaryons of Δcdc24 (supposed to exhibit reduced bccdc24 transcript levels) already show a strong phenotype: an inability to penetrate the host tissue, a significantly reduced growth rate and malformation of conidia, which tend to burst as observed for Δbcbem1. Also the formin BcSep1 has significant impact on hyphal growth and development, whereas the role of the putative ortholog of the yeast scaffold protein Far1 remains open: Δbcfar1 mutants have no obvious phenotypes.

Organisms are exposed to a tough environment, where acute daily challenges, like light, can strongly affect several aspects of an individual's physiology, including pathogenesis. While several fungal models have been widely employed to understand the physiological and molecular events associated with light perception, various other agricultural-relevant fungi still remain, in terms of their responsiveness to light, in the dark. The fungus Botrytis cinerea is an aggressive pathogen able to cause disease on a wide range of plant species. Natural B. cinerea isolates exhibit a high degree of diversity in their predominant mode of reproduction. Thus, the majority of naturally occurring strains are known to reproduce asexually via conidia and sclerotia, and sexually via apothecia. Studies from the 1970's reported on specific developmental responses to treatments with near-UV, blue, red and far-red light. To unravel the signaling machinery triggering development--and possibly also connected with virulence--we initiated the functional characterization of the transcription factor/photoreceptor BcWCL1 and its partner BcWCL2, that form the White Collar Complex (WCC) in B. cinerea. Using mutants either abolished in or exhibiting enhanced WCC signaling (overexpression of both bcwcl1 and bcwcl2), we demonstrate that the WCC is an integral part of the mentioned machinery by mediating transcriptional responses to white light and the inhibition of conidiation in response to this stimulus. Furthermore, the WCC is required for coping with excessive light, oxidative stress and also to achieve full virulence. Although several transcriptional responses are abolished in the absence of bcwcl1, the expression of some genes is still light induced and a distinct conidiation pattern in response to daily light oscillations is enhanced, revealing a complex underlying photobiology. Though overlaps with well-studied fungal systems exist, the light-associated machinery of B. cinerea appears more complex than those of Neurospora crassa and Aspergillus nidulans.

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.

Targeted gene inactivation is extensively used in the plant pathogenic fungus Botrytis cinerea for gene function analysis while strategies involving the expression of reporter genes have been rarely used due to the lack of appropriate expression vectors. Hence, an approach was initiated to establish an expression system for B.cinerea possessing the following features: (i) the targeted integration of constructs at defined gene loci which are dispensable under standard growth conditions, (ii) the use of promoter and terminator sequences allowing optimal gene expression, (iii) the use of codon-optimized reporter genes (Leroch et al., 2011), (iv) the use of multiple selection markers, and (v) the incorporation of a highly efficient cloning system. A set of basic vectors was generated by yeast recombinational cloning permitting a variety of protein fusions. The successful application of the expression system for labeling F-actin, the cytosol, the nuclei, the membrane, the ER and the peroxisomes was demonstrated. In addition, cloning vectors for bimolecular fluorescence complementation (BiFC) analyses for studying protein-protein interactions in situ were generated by splitting the codon-optimized gfp. The functionality of the constructed BiFC vectors was validated by demonstrating the interaction of the two white collar-like transcription factors BcWCL1 and BcWCL2 in the nuclei of growing B. cinerea hyphae., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

In the gray mold fungus Botrytis cinerea the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The ΔbccnA mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin-dependent genes and bccnA itself is positively affected by BcRcn1. Complementation of the Δbcrcn1 mutant with a GFP-BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the Ca(2+)/calcineurin-mediated signal transduction in B. cinerea, and that both proteins regulate fungal development and virulence.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops., Competing Interests: I have read the journal's policy and have the following conflicts: author Chinnappa Kodira currently works at 454 Life Sciences, Roche. All of the work reported in this manuscript was completed when he was in residence at the Broad Institute. None of the other authors have declared any competing interests.

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., (Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd. No Claim to Original US Government Works.)

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as alpha subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Deltabcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Deltabac, lesion development of Deltabcpka1 and DeltabcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Deltabac, Deltabcpka1 and DeltabcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Deltabcpka1 and DeltabcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Galpha subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.

Recently, we showed that the alpha subunit BCG1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin in the gray mold fungus Botrytis cinerea. To identify the transcription factor acting downstream of BCG1 and calcineurin, we cloned the gene encoding the B. cinerea homologue of CRZ1 ("CRaZy," calcineurin-responsive zinc finger transcription factor), the mediator of calcineurin function in yeast. BcCRZ1 is able to partially complement the corresponding Saccharomyces cerevisiae mutant, and the subcellular localization of the green fluorescent protein-BcCRZ1 fusion product in yeast cells depends on the calcium level and calcineurin activity. Bccrz1 deletion mutants are not able to grow on minimal media and grow slowly on media containing plant extracts. Hyphal morphology, conidiation, and sclerotium formation are impaired. The cell wall and membrane integrity, stress response (extreme pH, H(2)O(2), Ca(2+), Li(+)), and ability of the hyphae to penetrate the intact plant surface are affected in the mutants. However, BcCRZ1 is almost dispensable for the conidium-derived infection of bean plants. The addition of Mg(2+) restores the growth rate, conidiation, and penetration and improves the cell wall integrity but has no impact on sclerotium formation or hypersensitivity to Ca(2+) and H(2)O(2). The expression of a set of recently identified BCG1- and calcineurin-dependent genes is also affected in DeltaBccrz1 mutants, confirming that this transcription factor acts downstream of calcineurin in B. cinerea. Since the Bccrz1 mutants still respond to calcineurin inhibitors, we conclude that BcCRZ1 is not the only target of calcineurin.

The Galpha subunit BCG1 is essential for pathogenicity of the grey mould fungus Botrytis cinerea. Several processes such as the transition from primary infection to secondary invasive growth and the production of the phytotoxin botrydial are regulated by BCG1 via a cAMP-independent pathway. Our recent finding that the botrydial biosynthesis genes belong to the group of Ca(2+)/calcineurin-dependent genes suggested for the first time a connection between this Galpha subunit and the calcineurin signalling pathway. To investigate whether this co-regulation of genes by BCG1 and calcineurin is a common feature, a cDNA macroarray approach was used to compare the gene expression pattern of the wild-type and the Deltabcg1 mutant, non-treated or treated with the calcineurin inhibitor cyclosporin A. We identified three sets of genes whose expression was regulated either by both BCG1 and calcineurin, or only by one of them. Among the BCG1/calcineurin-co-regulated genes, we found a new gene cluster coding for a yet unknown polyketide secondary metabolite. Furthermore, we show for the first time in a phytopathogenic fungus that the phospholipase C (BcPLC1) is a component of the BCG1- and calcineurin-dependent signalling pathway as several BCG1- and calcineurin-dependent genes were downregulated in bcplc1 knock-down mutants.

To gain new insights into the signalling mechanisms of the grey mouldBotrytis cinerea, which causes several pre- and post-harvest diseases on a variety of host plants, we cloned, sequenced and functionally characterized a gene,btp1, encoding a novel 391-amino acid transmembrane protein. The protein BTP1 shows similarity to the transmembrane protein pth11, which is essential for appressorium formation and successful colonization of plant tissue in the rice blast fungusMagnaporthe grisea. Analyses of the deduced amino acid sequence ofbtp1predicted a sevenα-helical transmembrane topology, which is known to be typical for G protein-coupled receptors (GPCRs) and therefore the protein is thought to play a role in mediation of extracellular signals to intracellular effectors. The gene is located next to the genebcgstIIencoding a new putative glutathione S-transferase, and both genes are transcribed in opposite directions from the same promoter. BcGSTII shows similarity to the glutathione S-transferase GSTII ofSchizosaccharomyces pombe, a protein thought to be involved in detoxification of several antifungal drugs. From the sequence similarity of BTP1 to GPCRs, and its expressionin planta, we suggested that it might play a role in mediation of plant signals and therefore in pathogenicity. However, targeted gene replacement ofbtp1did not result in a phenotype markedly affecting either pathogenicity or sensitivity to chemical stress when compared with the wild-type strain; however, the ten-fold dilution of conidial suspension used for the pathogenicity assay resulted in slight reduction of virulence. Visible symptom development of the mutants on bean plants was also different from the wild-type. The brownish ring, which appears at the margin of secondary lesions in wild-type infections, was brighter and almost absent inΔbtp1 mutants. Interestingly, deletion ofbtp1not only affected the expression of the physically linkedbcgstIIgene, but in addition the expression of the other two GST-encoding genes inB. cinereaforbcgstIwas down-regulated,bcgstIIwas slightly up-regulated andbcgstIIIwas strongly up-regulated in the mutant. [ABSTRACT FROM AUTHOR]