Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine

Abstract

Background: Species in the ascomycete fungal genus Cordyceps have been proposed to be the teleomorphs of Metarhizium species. The latter have been widely used as insect biocontrol agents. Cordyceps species are highly prized for use in traditional Chinese medicines, but the genes responsible for biosynthesis of bioactive components, insect pathogenicity and the control of sexuality and fruiting have not been determined.

Results: Here, we report the genome sequence of the type species Cordyceps militaris. Phylogenomic analysis suggests that different species in the Cordyceps/Metarhizium genera have evolved into insect pathogens independently of each other, and that their similar large secretomes and gene family expansions are due to convergent evolution. However, relative to other fungi, including Metarhizium spp., many protein families are reduced in C. militaris, which suggests a more restricted ecology. Consistent with its long track record of safe usage as a medicine, the Cordyceps genome does not contain genes for known human mycotoxins. We establish that C. militaris is sexually heterothallic but, very unusually, fruiting can occur without an opposite mating-type partner. Transcriptional profiling indicates that fruiting involves induction of the Zn2Cys6-type transcription factors and MAPK pathway; unlike other fungi, however, the PKA pathway is not activated.

Conclusions: The data offer a better understanding of Cordyceps biology and will facilitate the exploitation of medicinal compounds produced by the fungus.

Figure 1

Life cycle and phenotypic polymorphism of C. militaris. The round conidia (from a solid culture) or the bar shaped blastospores (from a liquid culture) were inoculated onto caterpillar pupa or rice medium and incubated for up to 60 days. The resulting fertile fruiting bodies have protruded perithecia that contain asci. The ejected linear ascospores fragment and germinate to produce secondary pear-shaped conidia under nutrient poor conditions, that is, micro-cycle conidiation. Both the ascospores and secondary conidia can infect caterpillars. Scale bar: 5 μl.

Figure 2

Comparative genomics analysis of three insect pathogens. (a) Functional classification and comparison of C. militaris (CCM), M. anisopliae (MAA) and M. acridum (MAC) proteins, showing that C. militaris has fewer genes in each category. Each circle represents the relative fraction of genes represented in each of the categories for each genome. (b) Reciprocal blast analysis of the predicted proteins among three insect pathogens. The cut-off E value is at ≤ 1e-5.

Figure 3

Comparative genomics and evolutionary analysis of C. militaris. Scatter plots of Blast score ratio (BSR) analysis of (a) C. militaris (CCM), M. anisopliae (MAA) and M. acridium (MAC) genomes, and (b) CCM, MAA and F. graminearum (FG) genomes. The numbers in red at the lower left corners are the percentages of C. militaris species-specific sequences and the numbers at the upper left or lower right are the percentages of lineage-specific genes between pairs of genomes. (c) A maximum likelihood phylogenomic tree constructed using the Dayhoff amino acid substitution model showing the evolutionary relationship of C. militaris with different fungal species. Three insect pathogens are highlighted by the green shading. (d) Distribution of paralogous gene numbers with different levels of nucleotide similarity in C. militaris and other fungi. MY, million years.

Figure 4

Comparative analysis of the C. militaris mating-type (MAT) locus. (a) Comparative analysis of the C. militaris MAT locus with those of sexually heterothallic and homothallic fungal species. Genes labeled in the same color have orthologous relationships. (b) Syntenic relationship of the MAT loci and their flanking regions between the three insect pathogens C. militaris (CCM), M. anisopliae (MAA) and M. acridum (MAC).

Figure 5

Fruiting body development, sexuality and mating-type analysis. (a-c) Chinese Tussah silkmoth pupae were inoculated with conidia from the C. militaris Cm01 strain and incubated for 14 days (a), 29 days (b) and 59 days (c) to produce nascent, mid-term and developmentally mature fruiting bodies. (d-g) The mature fruiting bodies of the Cm01 strain do not produce perithecia (d, e) but those of strain Cm06 are completely covered with protruded perithecia (f, g). (h) PCR examination of different strains (numbers labeled on the top) showed that strains Cm06, Pm36 and 80399 contain the MAT1-1-1, MAT1-1-2 and MAT1-2-1 genes while Cm01 and other strains lack the MAT1-2-1 gene. (i) PCR examination of 30 randomly selected single spore isolates from the hybrid strain Cm06 showed that only 2 out of 30 isolates contain the MAT1-2-1 gene.

Figure 6

Fruiting structures of different mating-type isolates. (a, b) Sterile fruiting bodies formed on caterpillar pupae after inoculation of MAT1-1 (a) and MAT1-2 (b) isolates acquired by single conidial spore isolation from a MAT1-1/MAT1-2 hybrid strain, Cm06. (c-e) Fertile fruiting structures formed on caterpillar pupae after inoculation of the mixed conidia of MAT1-1 (Cm01) and MAT1-2 (Cm06) at ratios of 1:9 (c), 1:1 (d) and 9:1 (e), respectively. The right panels represent close-up views of corresponding sterile (without protruded perithecia) or fertile (with protruded perithecia) fruiting bodies. After inoculation, the pupae were incubated at 22°C with a 12:12 hour light:dark cycle for 60 days.

Figure 7

Cordycepin analogues and the C. militaris adenine metabolic pathway. (a) The structures of cordycepin analogues. (b) The C. militaris adenine metabolic pathway. Abbreviations for different enzymes: ADA, adenosine deaminase; ADE, adenine deaminase; ADEK, adenylate kinase; ADK, adenosine kinase; ADN, adenosine nucleosidase; AMPD, AMP deaminase; APRT, adenine phosphoribosytransferase; DADK, deoxyadenylate kinase; DAK, deoxyadenosine kinase; NDK, nucleoside-diphosphate kinase; NT5E, 5'-nucleotidase; PK, pyruvate kinase; PNP, purine nucleoside phosphorylase; 3'-RNR, ribonucleotide triphosphate reductase. The red dashed lines show metabolic pathways present in other organisms but absent in C. militaris.

Figure 8

Phylogenetic and modular analysis of C. militaris polyketide synthases compared with those involved in the production of human mycotoxins. (a) A neighbor-joining tree showing the relationships of ketoacyl CoA synthase (KS) domain sequences. (b) Modulation and comparison of C. militaris PKSs with those involved in production of mycotoxins. The PKS-NRPS hybrid proteins CCM_04722, CCM_08261 and CCM_08018 are not included in the analysis. Domain definitions: ACP, acyl carrier protein domain; AT, acyltransferase domain; CYC, cyclase domain; DH, dehydratase domain; ER, enoyl reductase domain; KR, ketoreductase domain; MT, methyltransferase domain; TE, thioesterase domain. The accessions and references for different mycotoxins are provided in the Materials and methods.

Figure 9

Differential gene expression by C. militaris in association with fruiting structure formation or growth in a liquid medium. (a) Estimation of significantly up- and down-regulated genes between different samples. (b) Heat map of protein kinases associated with the mitogen-activated and cAMP-dependent protein kinase pathways at different developmental stages. (c) Heat map of the highly expressed transcription factors at different developmental stages. Genes with expression values > 100 transcripts per million tags (TPM) are also indicated in red. Annotation information for the genes is provided in Table S19 in Additional file 1. DEG, differentially expressed gene. FB1, FB2 and FB3 are associated with nascent, stalk formation and mature developmental stages shown in Figure 5a-c, respectively. The transcriptome of undifferentiated mycelia harvested from SDB was included as a reference for gene expression analysis.

Figure 10

Putative signal transduction pathways regulating fruiting body development in C. militaris. The dashed lines show the cAMP-dependent PKA pathway, which might not be involved in control of fruiting in C. militaris. The transcription data for different components are provided in Table S19 in Additional file 1. AC, adenylate cyclase; CaMK, calmodulin-dependent protein kinase; CDK, cyclin-dependent kinase; PLC, phospholiapse C; RGS, regulator of G protein signaling.