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. Fungi produce numerous “secondary metabolites” (SMs) that are not essential for life but can provide an advantage under natural conditions, e.g. in fungal-host interactions. Here, we conducted the most comprehensive analysis to date of secondary metabolism in fungi using Fusarium fujikuroi. This fungus causes “bakanae” disease of rice and is best known for its ability to produce gibberellins (GAs). We show that GA production is limited to F. fujikuroi and provides a selective advantage during infection of the preferred host plant rice. Generation and analysis of a high-quality de novo F. fujikuroi genome sequence combined with comparisons to six other Fusarium genomes revealed the presence of 45 mostly unknown SM gene clusters. We provide a broad spectrum of experimental data including epigenetic, transcriptional, proteomic and chemical product analyses under different nitrogen and pH conditions. Two of the SM clusters (PKS19 and NRPS31) are not present in any other sequenced fungal genome. In planta expression studies revealed that the otherwise silent PKS19 cluster is induced on rice, but not on maize, suggesting a specific role for the PKS19-derived product during rice infection. Together, our results demonstrate the tremendous potential of a single fungal species to produce a diversity of SMs that likely contributes to adaptation to environmental changes.

Fungi occupy diverse environments where they are constantly challenged by stressors such as extreme pH, temperature, UV exposure and nutrient deprivation. Nitrogen is an essential requirement for growth, and the ability to metabolize a wide variety of nitrogen sources enable fungi to colonize different environmental niches and survive nutrient limitations. Favored nitrogen sources, particularly ammonium and glutamine, are used preferentially, while the expression of genes required for the use of various secondary nitrogen sources is subject to a regulatory mechanism called nitrogen metabolite repression. Studies on gene regulation in response to nitrogen availability were carried out first in Saccharomyces cerevisiae, Aspergillus nidulans, and Neurospora crassa. These studies revealed that fungi respond to changes in nitrogen availability with physiological and morphological alterations and activation of differentiation processes. In all fungal species studied, the major GATA transcription factor AreA and its co-repressor Nmr are central players of the nitrogen regulatory network. In addition to growth and development, the quality and quantity of nitrogen also affects the formation of a broad range of secondary metabolites (SMs). Recent studies, mainly on species of the genus Fusarium, revealed that AreA does not only regulate a large set of nitrogen catabolic genes, but can also be involved in regulating production of SMs. Furthermore, several other regulators, e.g. a second GATA transcription factor, AreB, that was proposed to negatively control nitrogen catabolic genes by competing with AreA for binding to GATA elements, was shown to act as activator of some nitrogen-repressed as well as nitrogen-induced SM gene clusters. This review highlights our latest understanding of canonical (AreA-dependent) and non-canonical nitrogen regulation mechanisms by which fungi may regulate biosynthesis of certain SMs in response to nitrogen availability.

Summary: Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet‐like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in ΔFfvel1 microarray‐studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet‐like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross‐genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.

Summary: Fusarium secondary metabolites are structurally diverse, have a variety of activities and are generally poorly understood biosynthetically. The F. fujikuroi polyketide synthase gene bik1 was previously shown to be responsible for formation of the mycelial pigment bikaverin. Here we present the characterization of five genes adjacent to bik1 as encoding a putative FAD‐dependent monooxygenase (bik2), an O‐methyltransferase (bik3), an NmrA‐like protein (bik4), a Zn(II)₂Cys₆ transcription factor (bik5) and an MFS transporter (bik6). Deletion of each gene resulted in total loss or significant reduction of bikaverin synthesis. Expression studies revealed that all bik genes are repressed by high amounts of nitrogen in an AreA‐independent manner and are subject to a time‐ and pH‐dependent regulation. Deletion of the pH regulatory gene pacC resulted in partial derepression while complementation with a dominant active allele resulted in repression of bik genes at acidic ambient pH. Transcription of all bik genes in strains lacking bik1, bik2 or bik3 was essentially eliminated, while transcription of some bik genes was detected in strains lacking bik4, bik5 or bik6. Thus, bikaverin synthesis is regulated by a complex regulatory network. Understanding how different factors influence the synthesis of this model secondary metabolite will aid understanding secondary metabolism in general.

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding α subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the Gα_i class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.