Project description: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 described about 100 years ago as the causative agent of Bakanae (M-bM-^@M-^\foolish seedlingM-bM-^@M-^]) disease of rice. Apart from GAs, the fungus is known to produce pigments and mycotoxins, but the biosynthetic genes are known for only eight products. 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-FTMS 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 polyketide synthase (PKS) and one non-ribosomal peptide synthetase (NRPS) gene cluster are unique for F. fujikuroi. Their products were identified by combining overexpression of cluster genes with HPLC-FTMS-based 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 at the transcriptional, translational and epigenetic levels. Examination of 3 different histone modifications, with 2 growth conditions for one of the modifications (Total of 4 samples)
Project description:Plants often generate secondary metabolites with antifungal properties as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier of antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocalgates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a translation initiation factor (eIF). De novo transcriptome assembly of the fungus revealed that eIF4A, a molecular target of rocaglates, replaces a critical amino acid in the rocaglate binding site. Moreover, genome-wide ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered Colletotrichum orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.
Project description:Genetic and molecular evidence to support the hypothesis that fungal secondary metabolites play a significant role in protecting the fungi against fungivory is scarce. We investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of the Collembola Folsomia candida feeding on mixed vs. single diets. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ΔLaeA) were combined with the high quality fungus Cladosporium cladosporioides as mixed diets or offered as single diets. We hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ΔlaeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ΔlaeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory.
Project description:Botrytis cinerea, the causal agent of gray mold and noble rot, is a phytopathogenic fungus with a scientific and economic relevance due to its effects in agriculture. This fungus employs various molecular tools, such as enzymes and secondary metabolites, to infect and colonize plant tissues. Pathogenicity and virulence factors are identified, highlighting their role in the infectious cycle. Our approach presents the description of a new infection mechanism in the phytopathogenic fungus Botrytis cinerea, based on the production of bioactive peptides. Those peptides were isolated from cultures with glucose (constitutive stage) and tomato (virulence inductor), and showed significant effects on virulence, suggesting a crucial function in the fungus-plant interaction. Biochemical and genetic analysis methods were employed to determine the expression of genes involved in peptide production, highlighting the potential for de novo peptide synthesis and degradation of parental proteins. The results indicate that B. cinerea has significant biotechnological potential, as it can generate peptides of interest for industrial and pharmaceutical applications. This work is pioneering in describing the peptidome of a phytopathogenic fungus and suggests new strategies for controlling microbial infections.
Project description: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 described about 100 years ago as the causative agent of Bakanae (“foolish seedling”) disease of rice. Apart from GAs, the fungus is known to produce pigments and mycotoxins, but the biosynthetic genes are known for only eight products. 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-FTMS 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 polyketide synthase (PKS) and one non-ribosomal peptide synthetase (NRPS) gene cluster are unique for F. fujikuroi. Their products were identified by combining overexpression of cluster genes with HPLC-FTMS-based 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 at the transcriptional, translational and epigenetic levels.
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.
Project description:Genetic and molecular evidence to support the hypothesis that fungal secondary metabolites play a significant role in protecting the fungi against fungivory is scarce. We investigated the impact of fungal secondary metabolites on transcript regulation of stress related expressed sequence tags (ESTs) of the Collembola Folsomia candida feeding on mixed vs. single diets. Aspergillus nidulans wildtype (WT; Ascomycota) able to produce secondary metabolites including sterigmatocystin (ST) and a knockout mutant with reduced secondary metabolism (A. nidulans ?LaeA) were combined with the high quality fungus Cladosporium cladosporioides as mixed diets or offered as single diets. We hypothesized that (i) A. nidulans WT triggers more genes associated with stress responses compared to the A. nidulans ?laeA strain with suppressed secondary metabolism, (ii) C. cladosporioides causes significantly different transcript regulation than the A. nidulans strains ?laeA and WT, and (iii) mixed diets will cause significantly different transcript expression levels than single diets. All three hypotheses are generally supported despite the fact that many functions of the affected ESTs are unknown. The results bring molecular evidence for the existence of a link between fungal secondary metabolites and responses in springtails supporting the hypothesis that fungal secondary metabolites act as a shield against fungivory. Twenty-three day old Folsomia candida were fed ad libitum for five days to fungal cuts respectively Cladosporium cladosporoides, Aspergillus nidulans WT, Aspergillus nidulans ?LaeA and two mixed diets of C.cladosporoides/A. nidulans WT (mix 1) and C. cladosporoides/A. nudlans ?LaeA (mix2) respectively. Four biological replicates were used for every treatment and a dye swap was used with the Cy3/Cy5 labels. This resulted in 20 samples which were analysed in 10 hybridisations executed in an interwoven loop design. The C. cladosporoides diet was used as the reference in the data analysis.
Project description:Contamination with enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem but there is no effective therapy available for EHEC infection. Biofilm formation is closely related with EHEC infection and is one of the mechanisms of antimicrobial resistance. Antibiofilm screening of 560 plant secondary metabolites against EHEC shows that ginkgolic acids C15:1 and C17:1 at 5 μg/ml and Ginko biloba extract at 100 μg/ml significantly inhibited EHEC biofilm formation on the surface of polystyrene, nylon membrane, and glass. Importantly, the working concentration of ginkgolic acids and G. biloba extract did not affect bacterial growth and has been known to be non-toxic to human. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, which were corroborated by reduced fimbriae production and biofilm reduction in EHEC. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of commensal E. coli K-12 strain. The current study suggests that plant secondary metabolites are important resource of biofilm inhibitors, as well as other bioactive compounds.
Project description:Naturally occurring Antisense Transcripts (NATs) compose an emerging group of regulatory RNAs. These regulatory elements appear in all organisms examined, but little is known about global expression of NATs in fungi. Analysis of currently available EST sequences suggests that 352 cis NATs are present in Aspergillus flavus. An Affymetrix GeneChip® microarray containing probes for these cis NATs, as well as all predicted genes in A. flavus, allowed a whole genome expression analysis of these elements in response to two ecologically important temperatures for the fungus. RNA expression analysis showed that 32 NATs and 2709 genes were differentially expressed between 37°C, the optimum temperature for growth, and 28°C, the conducive temperature for the biosynthesis of aflatoxin (AF) and many other secondary metabolites. These NATs correspond to sense genes with diverse functions including transcription initiation, carbohydrate processing and binding, temperature sensitive morphogenesis, and secondary metabolism. This is the first report of a whole genome transcriptional analysis of NAT expression in a fungus and shows that although less than 10% of putative NATs are differentially expressed in response to temperature, some of the NAT-cis gene interactions are likely to be important. Keywords: temperature response 3 samples (reps) for each temperature treatment were analyzed with a total of 6 samples.