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CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

ABSTRACT: Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS² experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

SUBMITTER: Hicks C

PROVIDER: S-EPMC10381663 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Hicks Carmen C Witte Thomas E TE Sproule Amanda A Hermans Anne A Shields Samuel W SW Colquhoun Ronan R Blackman Chris C Boddy Christopher N CN Subramaniam Rajagopal R Overy David P DP

Journal of fungi (Basel, Switzerland) 20230623 7

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely ass ...[more]

PMID: 37504684

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Project description:The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the "cryptic genome", specifically secondary metabolite expression.

Dataset Information

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Publications

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm <i>Fusarium graminearum</i> C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

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