Structure Elucidation of New Fusarins Revealing Insights in the Rearrangement Mechanisms of the <i>Fusarium</i> Mycotoxin Fusarin C

Fusarin C is a <i>Fusarium</i> mycotoxin that rearranges under reversed phase chromatographic conditions. In this study, the rearrangement of fusarin C was examined in detail, and the formation of fusarins under different conditions was optimized. All relevant fusarins including (10<i>Z</i>)-, (8<i>Z</i>)-, and (6<i>Z</i>)-fusarin C were isolated and identified by NMR. To confirm the involvement of the 2-pyrrolidone ring in the rearrangement of fusarin C, 15-methoxy-fusarin C was synthesized. For the first time, the structure of open-chain fusarin C was elucidated, and on the basis of these data, the rearrangement product of fusarin C was identified as <i>epi</i>-fusarin C. The results were confirmed by detailed NMR measurements and density functional theory calculations. Furthermore, a new fusarin C like metabolite, which was named dihydrofusarin C, was detected by analysis of the crude extract of fusarin C with high-performance liquid chromatography coupled to UV and Fourier transform mass spectrometry

Combining Mass Spectrometric Metabolic Profiling with Genomic Analysis: A Powerful Approach for Discovering Natural Products from Cyanobacteria

An innovative approach was developed for the discovery of new natural products by combining mass spectrometric metabolic profiling with genomic analysis and resulted in the discovery of the columbamides, a new class of di- and trichlorinated acyl amides with cannabinomimetic activity. Three species of cultured marine cyanobacteria, <i>Moorea producens</i> 3L, <i>Moorea producens</i> JHB, and <i>Moorea bouillonii</i> PNG, were subjected to genome sequencing and analysis for their recognizable biosynthetic pathways, and this information was then compared with their respective metabolomes as detected by MS profiling. By genome analysis, a presumed regulatory domain was identified upstream of several previously described biosynthetic gene clusters in two of these cyanobacteria, <i>M. producens</i> 3L and <i>M. producens</i> JHB. A similar regulatory domain was identified in the <i>M. bouillonii</i> PNG genome, and a corresponding downstream biosynthetic gene cluster was located and carefully analyzed. Subsequently, MS-based molecular networking identified a series of candidate products, and these were isolated and their structures rigorously established. On the basis of their distinctive acyl amide structure, the most prevalent metabolite was evaluated for cannabinomimetic properties and found to be moderate affinity ligands for CB₁

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

<div><p>The fungus <i>Fusarium fujikuroi</i> 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 <i>F. fujikuroi</i> 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 <i>F. fujikuroi</i> 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 <i>Fusarium</i>. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to <i>F. fujikuroi</i>, 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 (<i>PKS19</i>) and another that includes a non-ribosomal peptide synthetase gene (<i>NRPS31</i>) are unique to <i>F. fujikuroi</i>. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered <i>F. fujikuroi</i> strains overexpressing cluster genes. <i>In planta</i> expression studies suggest a specific role for the <i>PKS19</i>-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 <i>F. fujikuroi</i> as a rice pathogen.</p></div

Functional characterization of the PKS19 cluster, a putative polyketide biosynthetic gene cluster that is unique to <i>F. fujikuroi</i>.

<p>(A) Position and organization of the PKS19 gene cluster on <i>F. fujikuroi</i> chromosome VIII, GC content, distribution of active histone marks, and gene expression in the PKS19 cluster. Histone marks are described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g003" target="_blank">Figure 3</a>. Expression data are from microarray analysis of wild-type <i>F. fujikuroi</i> (strain IMI58289). Values are log₂ change in expression in a high versus low-nitrogen medium. H3K9ac and gene expression are correlated, as both are increased in the high-nitrogen medium. Some genes exhibited increased levels of H3K4me2 in the high-nitrogen medium, which also suggests transcription. (B) Chemical analysis of the SM product(s) of the PKS19 cluster. The traces show the combined extracted ion chromatograms for metabolites with molecular formulas [C₁₂H₁₆O₄+H]⁺, [C₁₂H₁₈O₅+H]⁺ and [C₁₂H₁₈O₄+H]⁺ determined by HPLC-FTMS of culture fluids from <i>F. fujikuroi</i> strains: IMI58289, wild-type strain; OE::TF, a strain over-expressing the transcription factor encoded by FFUJ_12242; OE::PKS19, a strain over-expressing the PKS19 gene FFUJ_12239; and OE::TF/OE::PKS19, a strain over-expressing both FFUJ-12242 and FFUJ_12239. (C) Northern blot analysis of PKS19 cluster genes in strains IMI58289 (WT), OE::TF, OE::PKS19 and OE::TF/OE::PKS19. (D) UV spectra of metabolites corresponding to peaks 1 through 4 from chromatograms shown in C. The similar spectra of the metabolites suggest structural similarity.</p

Whole genome comparison of <i>F. fujikuroi</i> with<i>F. verticillioides</i>.

<p>Dotplot of <i>F. fujikuroi</i> chromosomes and scaffolds against <i>F. verticillioides</i> calculated using MUMer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Delcher1" target="_blank">[121]</a> highlights overall collinearity. Orthologous DNA is represented by red dots, inverted segments are shown as blue dots. Inset magnifies <i>F. fujikuroi</i> chromosome XII, which has no homologue in the <i>F. verticillioides</i> scaffold set. The missing subtelomeric regions of chromosome IV in <i>F. fujikuroi</i> are highlighted by vertical purple lines. Dots that are located above or below the line indicating collinearity represent largely repetitive DNA.</p

Expression pattern^a of the secondary metabolite biosynthetic gene clusters under four growth conditions.

a<p>+++, >90% of the genes belonging to the cluster are expressed under the condition indicated. ++, 50–90% of the genes belonging to the cluster are expressed under the condition indicated. +, 25–50% of the genes belonging to the cluster are expressed under the condition indicated. −, 0–25% of the genes belonging to the cluster are expressed under the condition indicated.</p>b<p>DTC and STC indicate diterpene synthase and sesquiterpene synthase, respectively.</p><p>Key enzymes of which the respective product is known are indicated in bold letters and the respective metabolites are listed; n/k indicates that the corresponding metabolite is not yet known. Red labeled key enzymes and corresponding metabolites are <i>Fusarium fujikuroi</i>-specific.</p

Characterization of <i>F. fujikuroi</i> chromosomes and variation in acetylation and methylation statues of histone H3.

<p><b>A</b>: Information for chromosomes I, V and VIII is shown as examples of the 12 <i>F. fujikuroi</i> chromosomes (see<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475.s003" target="_blank">Figure S3</a> for additional chromosomes). For each chromosome, the position of the centromere is shown at the top; below this in descending order are: GC content, location of SM biosynthetic gene clusters, acetylation and methylation states of histone H3, and changes in gene expression. Variation in histone H3 modification statues indicates chromosomal regions in which genes are expressed (H3K9ac and H3K4me2) or silent (H3K9me3). “Δ expression up” indicates a more than twofold increase in gene expression during growth of <i>F. fujikuroi</i> in nitrogen-rich medium, whereas “Δ expression down” indicates an at least twofold decrease in gene expression. SM biosynthetic gene cluster locations are indicated by arrows labeled with the PKS, NRPS or TC (DTC means diterpene cyclase; STC means sesquiterpene cyclase) gene in each cluster (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-t004" target="_blank">Table 4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475.s020" target="_blank">Table S4</a>). For the same analyses of other <i>F. fujikuroi</i> chromosomes, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475.s003" target="_blank">Figure S3</a>. <b>B</b>: Immunocytological analysis of histone acetylation and methylation in <i>F. fujikuroi</i>. Detection of specific histone markers was performed with H3K9me3 and H3K9ac-specific antibodies. DNA was counterstained with DAPI. H3K9me3 is significantly enriched in heterochromatin that forms several chromocenters, while H3K9ac is evenly distributed in the nuclei (scale bar = 5 µm).</p

Relative expression of <i>PKS19</i> and <i>APS1</i> genes in maize and rice roots.

<p>Maize and rice roots were infected with <i>Fusarium fujikuroi</i> spores, and every 2 days RNA was isolated from three or five plants and used in real time PCR analysis. The expression levels were obtained using the delta-delta Ct and were normalized against three reference genes encoding a related actin (FFUJ_05652), a GDP-mannose transporter (FFUJ_07710) and ubiquitin (FFUJ_08398). The expression levels of <i>PKS19</i> (A) at 4 days in maize was arbitrarily set as 1, and all other expression levels were reported relative to it. In the case of the <i>APS1</i> gene (B), the expression levels of this gene at 4 days in maize was arbitrarily set as 1, and all other expression levels were reported relative to it.</p

Location of the NRPS/APS biosynthetic gene cluster on <i>F. fujikuroi</i> chromosome I, levels of histone modifications and gene expression within and flanking the cluster, and production of metabolites following overexpression of cluster genes <i>APS2</i> and <i>APS8</i>.

<p><b>A</b>: Synteny between the apicidin gene cluster in <i>F. semitectum </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Han2" target="_blank">[105]</a> and the apicidin-like gene cluster in <i>F. fujikuroi</i>. <b>B</b>: Histone modifications and gene expression in and flanking the cluster. Histone marks are described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g003" target="_blank">Figure 3</a>. Expression data were derived from microarray experiments in low and high nitrogen and are plotted as the changes in log₂ expression values in high-nitrogen medium compared to low-nitrogen medium. H3K9ac and gene expression are overall correlated, as both are increased under high nitrogen conditions. In some genes increased H3K4me2 was observed, also suggesting transcription. <b>C</b>: Chemical analysis of the product of the unique PKS19 gene cluster. The traces show the extracted ion chromatograms for [C₃₄H₄₈O₆N₅+H]⁺ (first line) and [C₃₅H₄₂O₇N₅+H]⁺ (second to fourth line) determined by HPLC-FTMS of an apicidin standard (first line) and of culture fluids from <i>F. fujikuroi</i> IMI58289, OE::APS8 and the OE::APS2/OE::APS8 mutant. <b>D</b>: UV spectra of apicidin and the apicidin-like compound. The similar spectra suggest a structural similarity.</p

Phylogenetic and phenotypic characteristics of <i>F. fujikuroi</i>.

<p><b>A</b>: Maximum likelihood tree showing phylogenetic relationships of <i>F. fujikuroi</i> and other species representing the Asian, African and American clades of the <i>Gibberella fujikuroi</i> complex (GFC), as well as <i>F. oxysporum</i>, <i>F. graminearum</i> and <i>F. solani</i>. The midpoint rooted tree is based on concatenated nucleotide sequences of 28 genes involved in primary metabolism that are highly homologous in different fusaria. Branches show bootstrap values (%), scale bar indicates amino acid substitutions per site. <b>B</b>: Phenotypic characteristics: a) Variation in pigmentation of the wild-type <i>F. fujikuroi</i> grown in a liquid medium containing (from top to bottom) 60 mM glutamine (fusarins), 6 mM NaNO₃ (fusarubins) and 6 mM glutamine (bikaverin); b) Perithecia resulting from a sexual cross of two isolates of <i>F. fujikuroi</i> with opposite mating types. Fusarubins account for the dark color of perithecia <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Studt1" target="_blank">[15]</a>; c) <i>F. fujikuroi</i> grown on complete medium and regeneration medium; d) fluorescent microscopy image of the DsRed-labeled <i>F. fujikuroi</i> wild type penetrating rice root cells during infection; e) Microscopic image of microconidial chain on KCl agar medium; f) characteristic symptoms of bakanae disease due to wild-type-infected rice seedlings (left) compared to the GA-deficient mutant (right).</p