Omics data reveal the unusual asexual-fruiting nature and secondary metabolic potentials of the medicinal fungus Cordyceps cicadae

Abstract

Background: Ascomycete Cordyceps species have been using as valued traditional Chinese medicines. Particularly, the fruiting bodies of Cordyceps cicadae (syn. Isaria cicadae) have long been utilized for the treatment of chronic kidney disease. However, the genetics and bioactive chemicals in this fungus have been largely unexplored.

Results: In this study, we performed comprehensive omics analyses of C. cicadae, and found that, in contrast to other Cordyceps fungi, C. cicadae produces asexual fruiting bodies with the production of conidial spores instead of the meiotic ascospores. Genome sequencing and comparative genomic analysis indicate that the protein families encoded by C. cicadae are typical of entomopathogenic fungi, including the expansion of proteases and chitinases for targeting insect hosts. Interestingly, we found that the MAT1-2 mating-type locus of the sequenced strain contains an abnormally truncated MAT1-1-1 gene. Gene deletions revealed that asexual fruiting of C. cicadae is independent of the MAT locus control. RNA-seq transcriptome data also indicate that, compared to growth in a liquid culture, the putative genes involved in mating and meiosis processes were not up-regulated during fungal fruiting, further supporting asexual reproduction in this fungus. The genome of C. cicadae encodes an array of conservative and divergent gene clusters for secondary metabolisms. Based on our analysis, the production of known carcinogenic metabolites by this fungus could be potentially precluded. However, the confirmed production of oosporein raises health concerns about the frequent consumption of fungal fruiting bodies.

Conclusions: The results of this study expand our knowledge of fungal genetics that asexual fruiting can occur independent of the MAT locus control. The obtained genomic and metabolomic data will benefit future investigations of this fungus for medicinal uses.

Keywords: Asexual fruiting; Bioactive metabolites; Cordyceps cicadae; Genomics; Mating type; Secondary metabolism.

Fig. 1

Phenotypes and development of asexual fruiting in C. cicadae. a Field collected fruiting bodies of C. cicadae developed on the cadaver of cicada Platylomia sp. b-d Fruiting body production of C. cicadae on the pupae of Chinese tussah silkworm (Antheraea pernyi) after inoculation for different times (labeled in each panel). Dpi, days post inoculation. e Fruiting bodies of C. cicadae produced on the rice medium 23 days post inoculation. f Asexual conidial spores produced on the fruiting bodies (arrows in the panels A, D and E). CO, conidium; Bar, 5 μm

Fig. 2

Genome structure comparison between C. cicadae and other closely-related fungi. a Scatter plots of Blast score ratio analysis of C. cicadae showing that the fungus is more closely related to I. fumosorosea than to C. militaris. b Dot blot analysis of C. cicadae, I. fumosorosea and C. militaris using ordered scaffold data. The blue spots show reverse-oriented relationships between the genomes. c Syntenic analysis of the representative scaffold structures between the three fungi. ISF, I. fumosorosea; CCAD, C. cicadae; CCM, C. militaris

Fig. 3

Structure, gene expression and loss-of-function analyses of the mating-type loci. a Structure comparison of the idiomorphic region between different fungi. In contrast to the structures of heterothallic or homothallic fungi, the MAT1-2 type of C. cicadae contains a truncated MAT1-1-1 gene. b Structure comparison of the MAT1-1-1 protein between different fungi. The truncated MAT1-1-1 encoded in the MAT1-2 type of C. cicadae has lost the N-terminus α-box domain. The MAT1-1-1 (AKV94677) from a MAT1-1 type of C. cicadae has a structure similar to those of C. militaris and B. bassiana. c RT-PCR analysis of MAT genes expressed by C. cicadae at different developmental stages. The mycelia harvested from the SDB (3 dpi), and primordia (13 dpi) and stroma (23 dpi) produced on silkworm pupae were used for RNA extraction and gene expression analysis. d Fruiting body production by the WT and mutant strains. Conidia of the WT and mutant strains were injected in the tussah silkworm pupae for 25 days

Fig. 4

Fungal secondary metabolisms. a Comparison of the core genes involved in secondary metabolisms between C. cicadae (CCAD) and other closely-related fungi. ISF, I. fumosorosea; CCM, C. militaris; CCO, C. confragosa (anamorph: Lecanicillium lecanii); BBO, B. brongniartii; BBA, C. bassiana. b Venn diagram analysis of the secondary metabolic gene clusters between C. cicadae and other fungi. c Differential expression of the core secondary metabolic genes in C. cicadae at different developmental stages. PCA plotting based on metabolomic data obtained from the positive (d) and negative ion mode (e) of LC-MS analysis. There were five independent repeats for each sample. Myc, mycelia grown in SDB for seven days; Pri, primordia harvested from the mycosed tussah silkworm pupae 13 days post injection; Str, stroma harvested from the mycosed silkworm pupae 22 days post injection

Fig. 5

Conservation and metabolite production analysis of the gene clusters involved in biosynthesis of oosporein and beauvericin in C. cicadae. a Schematic map of the oosporein biosynthetic gene cluster in C. cicadae and B. bassiana. b HPLC verification of oosporein (red peak) production in C. cicadae and B. bassiana. c Schematic map of the beauvericin biosynthetic gene cluster in different fungi. d HPLC analysis of beauvericin (red peak) production in different fungi. CCAD, C. cicadae; BBA, B. bassiana; ISF, I. fumosorosea. Myc, mycelia harvested from SDB broth; Pri, primordia; Str, stroma