Comparative Genomics and Transcriptomics During Sexual Development Gives Insight Into the Life History of the Cosmopolitan Fungus Fusarium neocosmosporiellum

Abstract

Fusarium neocosmosporiellum (formerly Neocosmospora vasinfecta) is a cosmopolitan fungus that has been reported from soil, herbivore dung, and as a fruit- and root-rot pathogen of numerous field crops, although it is not known to cause significant losses on any crop. Taking advantage of the fact that this species produces prolific numbers of perithecia in culture, the genome of F. neocosmosporiellum was sequenced and transcriptomic analysis across five stages of perithecium development was performed to better understand the metabolic potential for sexual development and gain insight into its life history. Perithecium morphology together with the genome and transcriptome were compared with those of the plant pathogen F. graminearum, a model for studying perithecium development. Larger ascospores of F. neocosmosporiellum and their tendency to discharge as a cluster demonstrated a duality of dispersal: the majority are passively dispersed through the formation of cirrhi, while a minority of spores are shot longer distances than those of F. graminearum. The predicted gene number in the F. neocosmosporiellum genome was similar to that in F. graminearum, but F. neocosmosporiellum had more carbohydrate metabolism-related and transmembrane transport genes. Many transporter genes were differentially expressed during perithecium development in F. neocosmosporiellum, which may account for its larger perithecia. Comparative analysis of the secondary metabolite gene clusters identified several polyketide synthase genes that were induced during later stages of perithecium development. Deletion of a polyketide synthase gene in F. neocosmosporiellum resulted in a defective perithecium phenotype, suggesting an important role of the corresponding metabolite, which has yet to be identified, in perithecium development. Results of this study have provided novel insights into the genomic underpinning of development in F. neocosmosporiellum, which may help elucidate its ability to occupy diverse ecological niches.

Keywords: Fusarium; mating type locus; perithecium; secondary metabolic genes; sexual development; transcriptome.

Figure 1

Perithecium development in F. graminearum (Fg) and F. neocosmosporiellum (Fn). (A) sexual stage definition. S1—ascogonium (arrowheads), S2—protoperithecia with conspicuous perithecial wall layers (arrows), S3—paraphyses (knuckle-like cells) released from squashed perithecium, S4—asci released from squashed perithecium, and S5—asci filled with ascospores. Scaling is the same for each stage in both species (Bar = 50 μm). (B) Ascus and ascospore morphology. (C) Mature perithecia grown on carrot agar. (D) Perithecium of Fg with cirrhus oozing from ostiole (arrow; left panel) and spore print from agar block containing numerous perithecia (right panel). (E) Perithecium of Fn with cirrhus oozing from ostiole (arrow; left panel) and spores fired in groups with visible halo of mucilage (right panel).

Figure 2

Mating type locus comparison (A) Structural organization of the MAT locus in F. graminearum and F. neocosmosporiellum. Genes that occur in both the MAT1-1 and MAT1-2 idiomorphs in heterothallic species occur together at a single MAT locus in these two homothallic species. In most ascomycetes, the MAT locus is flanked by APN2 (encoding a DNA lyase) and SLA2 (encoding a cytoskeletal protein), which are convergently transcribed toward the MAT genes. RNA-seq read coverage for MAT and flanking genes at S4 (ascus development stage) is plotted for both DNA strands (blue and green) with genome coordinates. Direction of transcription is the same for genes depicted as blue bars and opposite that of genes depicted as green bars. (B) MAT gene expression during perithecium development. Expression levels are presented as log₂[RPKM+1]; Blue lines: F. graminearum, Red lines: F. neocosmosporiellum.

Figure 3

Developmental stage-specific expression of CAZyme genes (A) Co-expressed groups of CAZyme genes in F. graminearum (Fg) and F. neocosmosporiellum (Fn) represented in trend plots of Z-score normalized RPKM values (y-axis) in a given group across perithecium development (x-axis). Gene numbers in parentheses. (B) Expression profiles of GH5, CE12, GH24, and GH25 CAZyme families identified in F. graminearum and F. neocosmosporiellum. Expression levels are presented as log₂[RPKM+1].

Figure 4

Conserved and divergent molecular events during perithecium development in F. graminearum (Fg) and F. neocosmosporiellum (Fn). (A) Differential expression (DE) analyses between two successive developmental stages [log₂[fold-change] > 1.5, false discovery rate (FDR) 5%]. Note increase in DE genes in ‘S2 vs. S3’ comparison in F. neocosmosporiellum. (B) Top five represented GO terms in functional enrichment analyses for DE genes upregulated in later stage. Red bars indicate values of negative log₁₀[adjusted P-value] for GO terms that are significantly enriched at 5% FDR. (C) Expression profiles of seven carbohydrate transporter genes differentially expressed and specifically induced in Fn at S3. Expression levels are presented as log₂[RPKM+1].

Figure 5

Secondary metabolism in F. neocosmosporiellum. (A) Herqueinone gene cluster consists of seven genes (phnA—phnG) in Penicillium herquei (top) compared with PKS35 (aka. PKSN) gene cluster in F. neocosmosporiellum (bottom). Percent amino acid sequence similarity of conserved genes is presented. Per-base coverage for RNA-seq reads is shown for both DNA strands (blue and green) during perithecium development (S0—S5). ADH, alcohol dehydrogenase; FMO, Flavin-dependent monooxygenase; OMT, O-methyltransferase; PKS, polyketide synthase; TF, transcription factor; ?: hypothetical protein (no predicted protein domain found in BLASTp search), Ψ: likely pseudogene (no expression). (B) Gene expression profiles of secondary metabolite biosynthetic genes during perithecium development. Four non-ribosomal peptide synthetases (NRPSs), 8 polyketide synthases (PKSs) and 3 terpene biosynthesis-related genes (TCs) are grouped according to their expression patterns, and presented as log₂[RPKM+1].

Figure 6

Knockout phenotypes of secondary metabolite biosynthesis genes induced at later stages of perithecium development. (A) Colony morphologies of deletion mutants (ΔPKS35, ΔPKS7, and ΔTC2) grown on potato dextrose agar [views of culture from above (left); from below (right)]. (B) Perithecium production on carrot agar (left). Asci released from a squashed perithecium (right). Note that ΔPKS7 produced protoperithecia but mature asci failed to develop. Bar = 100 μm.