Project description:The phytopathogenic fungus Fusarium fujikuroi is the causal agent of bakanae disease on rice due to its ability to produce gibberellins. Besides these phytohormones, F. fujikuroi is able to produce a wide range of other secondary metabolites (SMs), such as mycotoxins and pigments. Although much progress has been made in the field of secondary metabolism over the last years, the transcriptional regulation of SM biosynthetic genes is complex and far from being fully understood. Environmental conditions (e.g. nitrogen availability and pH), global and pathway-specific regulators as well as chromatin remodeling were shown to play major roles in this regulation. Here, the role of FfSge1, a homolog of the morphological switch regulators Wor1 and Ryp1 in Candida albicans and Histoplasma capsulatum, respectively, is explored with emphasis on secondary metabolism. FfSge1 is not required for conidia formation and pathogenicity, but is involved in vegetative growth. Genome-wide transcriptome analysis of the M-NM-^Tffsge1 deletion mutant compared to the wild type revealed that FfSge1 is a global regulator of secondary metabolism in F. fujikuroi that activates the expression of several SMs. In addition, FfSge1 is also required for expression of a yet uncharacterized SM gene cluster containing a noncanonical non-ribosomal peptide synthetase. Investigation of whole genome gene expression of the Fusarium fujikuroi wild type IMI58289, M-NM-^Tffsge1 mutant under nitrogen starvation and nitrogen sufficient conditions. In this study we hybridized in total 8 microarrays using total RNA recovered from a wild-type culture of F. fujikuroi IMI58289 and M-NM-^Tffsge1 mutant culture. All cultures were grown on a 6 mM Gln (10%) and a 60 mM Gln medium (100%). For each combination of culture and medium a biological replicate was created. Each chip measures the expression level of 14,397 genes from F. fujikuroi IMI58289 with eight 60-mer probes. Please note that the wild type samples have been published as part of the GEO accession GSE43745, but re-analyzed with the M-NM-^Tffsge1 mutant samples in the current study.

Project description:Histone modifications have been shown to be crucial for secondary metabolism in various filamentous fungi. Here we studied the influence of histone acetylation on secondary metabolite production in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites including pigments and mycotoxins. Deletion of the classical HDACs FfHdF1, FfHdF2 and FfHdF3 indicated that FfHdF1 and FfHdF2 are major regulators of secondary metabolism, whereas FfHdF3 is involved in developmental processes but dispensable for secondary metabolite production in F. fujikuroi. Microarray analysis with the major HDAC FfHdF2 revealed differential regulation of several secondary metabolite gene clusters, subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are responsible for gene silencing but also gene activation. Chromatin immunoprecipitation assays with M-NM-^TffhdF2 revealed significant alterations regarding the acetylation state in the landscape of secondary metabolite gene clusters thereby providing insights into the regulatory mechanism. In addition, the class I HDAC FfHdF1 also has major impact on secondary metabolism in F. fujikuroi. Furthermore, deletion of both ffhdF1 and ffhdF2 resulted in de-repression of secondary metabolites under normally repressing conditions. Thus, manipulation of HDAC encoding genes might provide a powerful tool for the activation of cryptic secondary metabolites. Investigation of whole genome gene expression of the Fusarium fujikuroi wild type IMI58289, M-NM-^TffhdF2 mutant under nitrogen starvation and nitrogen sufficient conditions. In this study we hybridized in total 12 microarrays using total RNA recovered from a wild-type culture of F. fujikuroi IMI58289 and M-NM-^TffhdF2 mutant culture. All cultures were grown on a 6 mM Gln (10%) and a 60 mM Gln medium (100%). For each combination of culture and medium a biological replicate was created. Each chip measures the expression level of 14,397 genes from F. fujikuroi IMI58289 with eight 60-mer probes.

Project description:Investigation of whole genome gene expression of the Fusarium fujikuroi wild type IMI58289 under gibberellin-inducing and -repressing conditions. Fusarium fujikuroi is a biotechnologically important fungus due to its almost unique ability to produce gibberellic acids (GAs), a family of phytohormones. The fungus was already described about 100 years ago as the causative agent of Bakanae (foolish seedling) disease of rice. Beside GAs, the fungus is known to produce some pigments and mycotoxins, but for only eight products the biosynthetic genes are known. Here we present a high-quality genome sequence of the first member of the Gibberella fujikuroi species complex (GFC), that allowed de novo genome assembly with 12 scaffolds corresponding to the 12 chromosomes. In this work, we focused on identification of all potential secondary metabolism-related gene clusters and their regulation in response to nitrogen availability by transcriptome, proteome, HPLC-FLPC and ChIP-seq analyses. We show that most of the cluster genes are regulated in a nitrogen-dependent manner, and that expression profiles fit to proteome and ChIP-seq data for some but not all clusters. Comparison with genomes of all available Fusarium species, including the recently sequenced F. mangiferae and F. circinatum, showed only a small number of common gene clusters and provides new insights into the divergence of secondary metabolism in the genus Fusarium. Phylogenetic analyses suggest that some gene clusters were acquired by horizontal gene transfer, while others were present in ancient Fusarim species and have evolved differently by gene duplications and losses. One PKS and one NRPS gene cluster are unique for F. fujikuroi. Their products were identified by combining overexpression of cluster genes with HPLC-FLPC -based product analyses. In planta, expression studies suggest a specific role of the PKS19 product in rice infection. Our results indicate that comparative genomics together with the used genome-wide experimental approaches is a powerful tool to uncover new secondary metabolites and to understand their regulation on the transcript, protein and epigenetic levels. In this study, we hybridized in total 15 microarrays using total RNA recovered from wild-type cultures of F. fujikuroi IMI58289. Two cultures were grown on a 6 mM Gln medium. Additionally, two technical replicates were created. Four cultures were grown on a 60 mM Gln medium. Again, two technical replicates were created. On a 6 mM NO3 medium, three cultures were grown, and two cultures on a 120 mM NO3 medium, with no technical replicates. Each chip measures the expression level of 14,397 genes from F. fujikuroi IMI58289 with eight 60-mer probes.

Project description:TCP transcription factors from the CYC2-class are involved in the development of monosymmetric flowers in all core eudicot species analysed so far. In Antirrhinum majus, the CYC2/TCP transcription factor CYCLOIDEA (CYC) is the molecular key regulator driving the development of flower monosymmetry (Luo D, Carpenter R, Vincent C, Copsey L, Coen E: Origin of floral asymmetry in Antirrhinum. Nature 1996, 383:794-799). In the Brassicaceae Iberis amara, a stronger expression of the CYC2 gene IaTCP1 in the small adaxial petals likely leads to the reduced petal size in comparison to large abaxial petals, with hardly any IaTCP1 expression. This results in the formation of the monosymmetric Iberis flower (Busch A, Zachgo S: Control of corolla monosymmetry in the Brassicaceae Iberis amara. PNAS 2007, 104:16714-16719). In contrast, the orthologous TCP/CYC2 transcription factor TCP1 from Arabidopsis thaliana, which forms equally sized and shaped petal pairs, only shows an early and transient expression in the adaxial area of floral primordia. This implies that monosymmetry in the Brassicaceae evolved through a heterochronic expression shift of the TCP/CYC2 key regulator gene IaTCP1. Transgenic Arabidopsis plants overexpressing IaTCP1 and TCP1 develop smaller petals whereas transgenic plants overexpressing CYC from Antirrhinum majus produce larger flowers. In any case, petal size is affected. To compare the effects of the three CYC2 TCP transcription factors on downstream (regulatory) networks in Arabidopsis thaliana, a microarray analysis was conducted. The coding sequences of the TCP/CYC2 transcription factors IaTCP1, TCP1 and CYC were cloned into the pBAR vector (GenBank: AJ251014), resulting in the constructs #0522 (IaTCP1), #0569 (TCP1) and #0577 (CYC). In pBAR, all genes are under the control of the CaMV35S-promoter. Arabidopsis plants were transformed (via floral dip) with respective constructs and also with the empty vector (pBar). Transgenic plants (T1) with petal size deviation from the control (plants transformed with the empty vector and wild type) were selfed and resulting T2 lines with petal size deviations from control were selected. Inflorescence buds from secondary inflorescences were harvested from transgenic T2 plants that formed smaller (#0255 or #0569) or larger (#0577) petals in the main inflorescence. Total RNA was isolated and sent to the Integrated Functional Genomics Service at the University of MÃ¼nster, Germany, which carried out probe preparation, hybridization and statistical analysis of the data. Differential gene expression was always determined from a comparison of gene expression from #0522, #0569 and #0577, respectively, against the control (#pBar; inflorescence gene expression in plants transformed with an empty vector).

Project description:Histone posttranslational modifications (HPTMs) are involved in regulating the synthesis of fungal bioactive compounds. The exact molecular mechanisms of the silencing/activation of secondary metabolism (SM) clusters by these epigenetic events however are not yet fully understood. This work applies a combined approach of quantitative mass spectrometry (LC-MS/MS) and chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) to identify the chromatin landscape in two metabolic states: primary and secondary metabolism. Furthermore, to link the particular chromatin states to the expression of condition specific genes, genome wide transcriptome (RNA-seq) was performed. Strikingly, we found that silent A. nidulans SM clusters are free of repressive H3K9me3 though this heterochromatic mark forms distinguished peaks flanking the many SM clusters. In addition, silent SM clusters do not contain detectable levels of activating histone marks such as H3K4me3, H3K36me3 or H3Ac, which, to some extent, are established upon activation of the clusters. In order to investigate the function of dynamic H3K4 methylation/demethylation in transcription, we characterized the KdmB- Jarid1 family histone demethylase. The in vitro assay using heterologously expressed KdmB showed that it is an active demethylase; moreover, MS/MS as well ChIP-seq approaches revealed that it targets H3K4me3 in vivo mediating transcriptional repression. KdmB positively regulates the expression of 40% of A. nidulans SM genes and this function appears to be independent of its demethylase activity. Our bioinformatics approach revealed two states of H3K4me3 in A. nidulans genome: loci with low levels of this mark are more disposed to differential expression in response to environmental clues, while the genes marked by high H3K4me3 levels are constitutively transcribed in our experimental conditions. Taken together our data reveal important role of H3K4 methylation/demethylation in transcription regulation. Furthermore, this study presents the first genome-wide map of H3K4me3, H3K9me3, H3K36me3 and H3Ac in A. nidulans in different metabolic conditions. Two strains, wild type and kdmB deletion, at two conditions, growth at primary (17h) and secondary (48h), were analysed. Each sample was replicated.

Project description:Histone posttranslational modifications (HPTMs) are involved in regulating the synthesis of fungal bioactive compounds. The exact molecular mechanisms of the silencing/activation of secondary metabolism (SM) clusters by these epigenetic events however are not yet fully understood. This work applies a combined approach of quantitative mass spectrometry (LC-MS/MS) and chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) to identify the chromatin landscape in two metabolic states: primary and secondary metabolism. Furthermore, to link the particular chromatin states to the expression of condition specific genes, genome wide transcriptome (RNA-seq) was performed. Strikingly, we found that silent A. nidulans SM clusters are free of repressive H3K9me3 though this heterochromatic mark forms distinguished peaks flanking the many SM clusters. In addition, silent SM clusters do not contain detectable levels of activating histone marks such as H3K4me3, H3K36me3 or H3Ac, which, to some extent, are established upon activation of the clusters. In order to investigate the function of dynamic H3K4 methylation/demethylation in transcription, we characterized the KdmB- Jarid1 family histone demethylase. The in vitro assay using heterologously expressed KdmB showed that it is an active demethylase; moreover, MS/MS as well ChIP-seq approaches revealed that it targets H3K4me3 in vivo mediating transcriptional repression. KdmB positively regulates the expression of 40% of A. nidulans SM genes and this function appears to be independent of its demethylase activity. Our bioinformatics approach revealed two states of H3K4me3 in A. nidulans genome: loci with low levels of this mark are more disposed to differential expression in response to environmental clues, while the genes marked by high H3K4me3 levels are constitutively transcribed in our experimental conditions. Taken together our data reveal important role of H3K4 methylation/demethylation in transcription regulation. Furthermore, this study presents the first genome-wide map of H3K4me3, H3K9me3, H3K36me3 and H3Ac in A. nidulans in different metabolic conditions.

Dataset Information

Knock-down of the methyltransferase Kmt6 reliefs H3K27me3 and induces expression of cryptic otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi [gene expression]

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