The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

ABSTRACTFusarium fujikuroiproduces a variety of secondary metabolites, of which polyketides form the most diverse group. Among these are the highly pigmented naphthoquinones, which have been shown to possess different functional properties for the fungus. A group of naphthoquinones, polyketides related to fusarubin, were identified inFusariumspp. more than 60 years ago, but neither the genes responsible for their formation nor their biological function has been discovered to date. In addition, although it is known that the sexual fruiting bodies in which the progeny of the fungus develops are darkly colored by a polyketide synthase (PKS)-derived pigment, the structure of this pigment has never been elucidated. Here we present data that link the fusarubin-type polyketides to a defined gene cluster, which we designatefsr, and demonstrate that the fusarubins are the pigments responsible for the coloration of the perithecia. We studied their regulation and the function of the single genes within the cluster by a combination of gene replacements and overexpression of the PKS-encoding gene, and we present a model for the biosynthetic pathway of the fusarubins based on these data.

Summary Filamentous fungi produce a vast array of secondary metabolites (SMs) and some play a role in agriculture or pharmacology. 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. One important regulatory layer in SM biosynthesis involves histone modifications that render the underlying genes either silent or poised for transcription. Here, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by trimethylated lysine 27 on histone 3 (H3K27me3). Kmt6, the methyltransferase responsible for establishing this histone mark, appears to be essential in this fungus, and knock-down of Kmt6 in the KMT6kd strain shows a drastic phenotype affecting fungal growth and development. Transcription of four so far cryptic and otherwise silent putative SM gene clusters was induced in the KMT6kd strain, in which decreased expression of KMT6 is accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, named STC5, was analysed in more detail thereby revealing a novel sesquiterpene.

In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn 2؉ -dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as ⌬ffhda1 ⌬ffhda2 mutants resemble the uninfected control plant. Microarray analysis with a ⌬ffhda1 mutant that has lost the major HDAC revealed differential expression of secondarymetabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the ⌬ffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.