Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates

Abstract

Background: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description.

Methods: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions.

Results: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed.

Conclusions: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Keywords: Accessory chromosomes; Apicidin; Biosynthetic gene clusters; Fungal plant pathogens; Fusarium poae; Genomics; Mass spectrometry; Metabolomics; Secondary metabolites.

Fig. 1

Genome assembly of Fusarium poae isolate Fp157 with predicted biosynthetic gene clusters (BGCs), centromeric regions and telomeric repeats overlaid. BGC sizes are not to scale. Asterisks indicate duplicated koraiol synthases with > 98% nt ID. Annotations above BGCs refer to associated synthase/synthetase clades, or associated mycotoxin products where known. RE indicates repeat element contents expressed as a percentage of each chromosome or contig length calculated independently of the rest of the genome (repeat content attributable to duplications between chromosomes was not calculated). Similarly, evidence of repeat-induced point mutation (RIP) was calculated independently per sequence and is expressed as the percent of each sequence predicted to be RIP-affected (evidenced by calculation of low GC-content compared to average dinucleotide frequencies). Predicted centromeres and telomeres were removed from all sequences prior to RIP analysis

Fig. 2

Consensus chemical phenotypes of 38 Canadian isolates of Fusarium poae. Heatmap values represent the frequency of detection for mass features averaged over five media conditions per isolate. Dendrograms at left and top generated from hierarchical cluster analysis of detection frequencies. Annotations: APS, apicidins and apicidin-like features; AUR, aurofusarin; BEA, beauvericin; DAN, diacetylnivalenol; DAS, diacetoxyscirpenol; F-X, fusarenon-X; FUS-assoc., fusarin-associated features; MAS, monoacetoxyscirpenol; NEO, neosolaniol/isoneosolaniol; NIV, nivalenol; RUB, rubrofusarin; SCR, scirpentriol; TRI-assoc., trichothecene-associated features other than those verified by comparison to commercial standards or published MS² spectra (see text); W-493, cyclodepsipeptides W-493 A/B

Fig. 3

(a) Apicidin (APS) subnetwork generated from feature-based molecular network analysis of APS-like signals using GNPS (release_23) [47], visualized in cytoscape. Nodes represent distinct features (peaks) with unique retention times and m/z, and are either connected by cosine similarity score (threshold = 0.7, blue line) or adduct identity match generated using IIN module [48] in MZMine2 [49] (red line). Nodes are coloured based on ion identity, and node outlines are coloured by annotation method: red annotations derive from top hit from in silico MS² structural prediction using Sirius / CSI Finger-ID [50], green annotations derive from spectral matching to GNPS database, grey outlines represent spectra whose adducts were annotated by manual inspection of raw data. Potentially novel APS-like signals are annotated with exact masses (< 5 ppm). Node size represents relative size of signal calculated by precursor intensity (sum of all spectra in MS² scan). (b) Mirror plot comparing MS² spectra of predicted APS and APS-G signals. Substructures are coloured based on association with m/z motifs: blue m/z occur in nearly all APS-associated mass feature MS² scans, purple fragments are detected in most spectra associated with tryptophan-bearing apicidins, red fragments correspond to predicted phenylalanine moiety-associated fragments and appear only in putative APS-G spectra. For detailed information see Additional file 11. (c) Synteny visualization of FpAPS gene cluster residing on putative accessory chromosome of Fp157 as compared to homologous cluster in F. incarnatum KCTC 16676 (Genbank accession GQ331953) [51]. Blue arrows are predicted genes, red squares are predicted transposable elements. Predicted APS gene functions: 1, NRPS; 2, transcription factor; 3, pyrroline reductase; 4, aminotransferase; 5, fatty acid synthase; 6, O-methyl transferase; 7, cytochrome P450; 8, cytochrome P450; 9, FAD-dependent oxidase; 10, short-chain reductase; 11, efflux pump; 12, reductase

Fig. 4

Gene synteny visualization of predicted BGCs on Fp157 core chromosomes and homologous regions on accessory chromosomes. (a) FpNRPS4-associated region on core chromosome 1 compared to homologous region on Contig_2. (b) FpPKS2-associated region on core chromosome 2 compared to homologous region on Contig_2

Fig. 5

Phylogenetic analysis of 4141 single-copy BUSCO orthologs from F. poae isolates investigated in this study including Belgian isolate 2516. F. venenatum assembly GCA_900,007,375.1 was included as an outgroup to determine the true location of root (double slashes indicate truncated branch leading to outgroup). Evolutionary histories were inferred using the Maximum Likelihood (ML) method. Branch lengths are measured in number of substitutions per site, and node values indicate Ultrafast bootstrapping values (n = 1000). Biosynthetic gene annotations: the FpAPS cluster and the disrupted FpNRPS4 (Ψ) synthetase are associated with accessory chromosome sequences in the Fp157 assembly, whereas the disrupted FpPKS2 (Ψ) synthase was assembled to a core chromosome in Fp157. Purple and green pie charts represent size of fragments detected relative to concatenated Fp157 FpNRPS4 (Ψ) or PKS2 (Ψ) sequences. Empty pie charts indicate absence of FpNRPS4 (Ψ) detection