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

ABSTRACT: Filamentous fungi produce a vast array of secondary metabolites (SMs) and some of them are applied in agriculture or pharmacology. Recent sequencing of the rice pathogen Fusarium fujikuroi revealed the presence of far more SM-encoding genes than known products. SM production is energy-consuming and thus tightly regulated, leaving the majority of SM gene clusters silent under laboratory conditions. It is now well established that one important regulatory layer in SM biosynthesis involves histone modifications that render the genes either silent or poised for transcription. In this study, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by H3K27me3. In this study, we performed comparative transcriptomics of a knock-down mutant of the responsible methyltransferase Kmt6 involved in H3K27 methylation grown on either solid complete medium or solid synthetic ICI medium. Overall four so far cryptic and otherwise silent putative SM gene clusters were significantly induced in the KMT6kd strain accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, the STC5 gene cluster, was analysed in detail and heterologous expression of the key enzyme allowed for the identification of the first pathway-specific intermediate (1R,4R,5S)-guaia-6,10(14)-diene.

ORGANISM(S): Fusarium fujikuroi

PROVIDER: GSE80477 | GEO | 2016/07/04

SECONDARY ACCESSION(S): PRJNA319026

REPOSITORIES: GEO

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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.

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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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