Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen Fusarium fujikuroi Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

Abstract

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.

Fusarium in the age of genomics

“…Proper assessment of the role of specific genetic elements such as telomeres, centromeres and repetitive elements will benefit from the availability of a fully assembled genome. To date, the best assembled genomes of Fusarium species are F. graminearum (King et al, 2015); F. fujikuroi (Wiemann et al, 2013) and F. poae (Vanheule et al, 2016). In addition, genome compartmentalization and structural rearrangements within and between chromosomes can only be studied accurately, when a genome is assembled to chromosome-sized contigs.…”

Two Histone Deacetylases, FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

“…A direct link between histone acetylation and secondary-metabolite gene expression in filamentous fungi was first shown for the aflatoxin gene cluster in Aspergillus parasiticus (13). Later on, an enrichment for acetylated histone 3 lysine 9 (H3K9ac), H3K14ac, or H4K12 was also found in the landscape of secondary-metabolite gene clusters under active transcription but was missing in a silencing environment in other fungi, including Fusarium fujikuroi (8,9,(14)(15)(16)(17). These findings suggest an important role of HATs and HDACs in the regulation of genes involved in secondary metabolism.…”

“…Besides gibberellins, the fungus produces a wide range of other secondary metabolites, such as the red polyketide pigments bikaverin (26,27) and fusarubins (28,29) and mycotoxins such as fusarin C (30), fusaric acid (31), or fumonisins (32). Furthermore, recent sequencing of the genome of F. fujikuroi wild-type (WT) strain IMI58289 revealed the presence of about 40 additional putative secondary-metabolite gene clusters, most of which are cryptic and silent under laboratory conditions (17). Possible strategies to awaken these clusters, thereby unraveling the respective products, include various growth conditions, overexpression of key enzymes and/or pathway-specific transcription factors, or modification of histonemodifying enzymes, which in turn leads to alterations of the surrounding chromatin landscape.…”

“…Plasmid construction for deletion of all three HDACs was accomplished by yeast recombinational cloning (37). Therefore, the 5= and 3= regions of the corresponding genes were amplified with appropriate primer pairs: for the 5= region, primers 5F and 5R were used, and for the 3= region, primers 3F and 3R were used, based on the available genomic sequence of F. fujikuroi IMI58289 (17). Hygromycin B was used as a resistance marker.…”

Pictet–Spengler reaction-based biosynthetic machinery in fungi

“…This is why natural products remain a common stimulating force both for synthetic and material chemistry (8)(9)(10), and for pharmaceutical and agrochemical industries (6,7,39). Concerning the microbial generation of natural products, the long-time evolution has enabled microbes to organize the biosynthetic machineries in an ingenious way so that they can produce the organic molecular diversity as case-dependent responses to various outside events such as symbiosis (40), gene transfer (41), and host invasion (42). To get more structurally unprecedented small molecules, it is urgently necessary to activate the silent or less-active biosynthetic pathways in the producing organisms.…”