Genome sequence of the insect pathogenic
fungus Cordyceps militaris, a valued traditional
chinese medicine
Zheng et al.
Zheng et al. Genome Biology 2011, 12:R116
http://genomebiology.com/2011/12/11/R116 (23 November 2011)
Zheng et al. Genome Biology 2011, 12:R116
http://genomebiology.com/2011/12/11/R116
RESEARCH Open Access
Genome sequence of the insect pathogenic
fungus Cordyceps militaris, a valued traditional
chinese medicine
Peng Zheng1?, Yongliang Xia1?, Guohua Xiao1?, Chenghui Xiong1, Xiao Hu1, Siwei Zhang1, Huajun Zheng2,
Yin Huang2, Yan Zhou2, Shengyue Wang2, Guo-Ping Zhao1,2, Xingzhong Liu3, Raymond J St Leger4 and
Chengshu Wang1*
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.
Background insect biocontrol agents [2,3]. Although C. militaris and
The Ascomycete genus Cordyceps includes over 500 Cordyceps sinensis (syn. Ophiocordyceps sinensis) are
species that are pathogens of arthropods. Cordyceps best known as traditional Chinese medicines, they are
militaris (CCM) is the type species and occurs through- also increasingly being studied and used in the West
out much of the Northern Hemisphere as a pathogen of [4,5]. An array of pharmacologically active components
lepidopteran insect pupae [1]. C. militaris is readily has been identified, including cordycepin, cordycepic
characterized by the sexual fruiting bodies forming on acids, polysaccharides and macrolides [6]. Cordycepin
mycosed pupae, the structures giving the fungus its (3?-deoxyadenosine) has so far only been reported in C.
common name of ?pupa grass? in China. Anamorphic militaris and is a broad spectrum antimicrobial [5] and
Cordyceps species, such as Beauveria spp., Metarhizium polyadenylation inhibitor that is currently undergoing
spp. and Paecilomyces spp., have been developed as clinical trials against cancers [7]. The biosynthetic path-
way of cordycepin production has not been determined.
* Correspondence: cswang@sibs.ac.cn In spite of their market values - for example, >
? Contributed equally $10,000 per kilo for the fruiting bodies of the un-culti-
1Key Laboratory of Insect Developmental and Evolutionary Biology, Institute vatable C. sinensis [8] - very little is known about sex
of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China and developmental processes in Cordyceps species, and
Full list of author information is available at the end of the article remedying this deficiency should help in production/
? 2011 Zheng et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
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cultivation of these enigmatic fungi. C. militaris is nota- pharmaceuticals underlying the widespread medical
ble as it readily performs sexual reproduction on artifi- impact of Cordyceps spp., and identify potential safety
cial media and is thus a good target for studying the hazards, including genes for known human mycotoxins.
molecular underpinnings of sex and development in
Cordyceps spp. (Figure 1). C. militaris also has the Results
potential to be a versatile new model for studying the Genome sequencing and general features
evolution of sex and reproductive structures. While cur- The C. militaris genome was shotgun sequenced to 147
rent fungal models have provided numerous insights ? coverage and assembled into 33 scaffolds with an N50
into the evolution of sex [9], there is still much to be of 4.6 Mb and a total genome size of 32.2 Mb. The gen-
understood about the mechanisms, evolution and ecolo- ome is smaller than either the broad host range Metar-
gical impact of sexuality in fungi. This is, in part, hizium anisopliae (MAA) or the locust-specific
because fungal mating and sexual cycles are often com- pathogen Metarhizium acridum (MAC) that we
plicated; for example, aspergilli have both self-fertile sequenced previously (Table 1). The characteristic telo-
(homothallism) and self-sterile (heterothallism) mating meric repeats (TTAGGG/CCCTAA)n were found at
systems [10]. either 5? or 3? terminal of 13 scaffolds, including the
There is also much to be learnt about the nature and terminal anchoring of two scaffolds, that is, the com-
evolution of interactions of Cordyceps spp. with their plete chromosomes. From mapping > 5,000 expressed
hosts and with the wider environment. As entomo- sequence tags [12], the C. militaris genome was esti-
pathogenicity appears to have evolved independently in mated to be > 99% complete. The genome was predicted
Cordyceps and two Metarhizium species [11], compara- to encode 9,684 protein genes, which is slightly fewer
tive genomics will provide independent assessments of than M. anisopliae and M. acridum (Table 1). Conse-
what is required to be entomopathogenic, identify the quently, many protein functional categories are smaller
degree to which evolution between these fungi has in Cordyceps than in Metarhizium spp. (Figure 2a).
been convergent, and identify the genomic basis of However, like M. anisopliae (17.6%) and M. acridum
their differing physiologies and host-specificity. Last (15.1%), C. militaris has a higher proportion of its genes
but not least, genomic sequencing of C. militaris will encoding putatively secreted proteins (15.9%) than other
enable a systematic exploration of the biology and sequenced ascomycetes (5 to 10%) [10,13,14].
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 ?l.
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Table 1 Comparison of genome features among three amino acid identity with either M. anisopliae or M. acri-
insect pathogens dum, slightly higher than with the plant pathogens F.
Features C. militaris M. anisopliae M. acridum graminearum (61.6%) and Magnaporthe oryzae (56.0%)
Size (Mb) 32.2 39.0 38.1 (Table 2). Thus, the three insect pathogenic fungi are
Coverage (fold) 147 ? 100 ? 107 ? more highly diverged than F. graminearum, Fusarium
Percentage G+C 51.4 51.5 50.0 oxysporum and Fusarium verticillioides, which share an
content average of 85% nucleotide sequence identity [14], and
Percentage repeat rate 3.04 0.98 1.52 Aspergillus nidulans, Aspergillus fumigatus and Aspergil-
Protein-coding genes 9,684 10,582 9,849 lus oryzae, which share an average of 68% amino acid
Gene density (genes 257 271 259 sequence identity [13], and Trichoderma reesei, Tricho-
per Mb) derma virens and Trichoderma atroviride, which share
Exons per gene 3.0 2.8 2.7 an average of > 70% amino acid sequence identity [15].
Percentage secreted 16.2 17.6 15.1 The regions containing at least three contiguous open
proteins
reading frames that are not present in the reference gen-
tRNA 136 141 122
ome are designated as genomic islands (GIs) [16].
Pseudogenes 102 363 440
Whole genome reciprocal analysis of three insect patho-
NCBI accession AEVU00000000 ADNJ00000000 ADNI00000000
gens demonstrated that, in comparison to Metarhizium
Mb, mega base pairs.
spp., C. militaris has 52 GIs (2% coverage of its genome,
harboring 21% of its species-specific genes), which is
An InterproScan analysis identified 2,736 conserved many more than M. anisopliae (8 GIs, 0.3%) or M. acri-
protein families in C. militaris (containing 6,725 pro- dum (5 GIs, 0.2%) when referenced to C. militaris. As
teins), fewer than those in M. anisopliae (7,556 proteins in aspergilli [17], many C. militaris species-specific
in 2,796 families) or M. acridum (6,948 proteins in gene-encoding proteins do not have conserved domains
2,746 families) [11]. In particular, the number of trans- and the genes are clustered together to form GIs (Table
posases is much fewer in C. militaris (4) than in Metar- 2). A phylogenomic analysis established that the Cordy-
hizium spp. (148 in M. anisopliae and 20 in M. ceps lineage is more closely related to the wheat patho-
acridum) or other sequenced ascomycetes (15 to 426) gen F. graminearum (divergence time of 200 to 260
(Table S1 in Additional file 1). The C. militaris genome million years ago (MYA)) than it is to Metarhizium spp.
lacks retrotransposase (Table S2 in Additional file 1), (26 to 34 MYA) (Figure 3c). Thus, the lineage leading to
and has more than three-fold fewer pseudogenes than C. militaris appears to have diverged from plant patho-
Metarhizium spp. (Table S3 in Additional file 1). About gens around the Triassic-Jurassic boundary (200 MYA),
16% of the predicted C. militaris genes (1,547) are puta- while M. anisopliae and M. acridum diverged after the
tively involved in pathogen-host interactions; this pro- Cretaceous Extinction Event (65 MYA) [18]. Analysis of
portion is slightly lower than for Metarhizium spp. paralogous genes found only one pair of C. militaris
(17.3% in MAA and 16.5% in MAC) but is higher than genes with > 90% nucleotide sequence similarities (Fig-
four plant pathogens (10.8 to 15.5%; P = 0.0476; false ure 3d), which is similar to Neurospora crassa (one pair)
discovery rate (FDR) = 0.0152) (Table S4 in Additional [13] and F. graminearum (two pairs) [19]. Analysis of 24
file 1). paired C. militaris genes showing > 70% nucleotide
More than 50% of M. anisopliae and M. acridum pro- identities found a strong overall C:G to T:A mutation
teins have > 90% identity [11]. Although the rarely bias (Figure S1 in Additional file 2), consistent with
observed sexual stages of Metarhizium spp. have been repeat-induced point mutations, the DNA methylation-
identified as a Cordyceps species [1], the analysis linked processes that cause mutations of repeated fungal
revealed that < 2% of C. militaris genes were highly con- sequences [15,20].
served in comparison with those from Metarhizium
spp., that is, had Blast score ratio (BSR) values close to Protein family analysis
1 (Figure 3a). A similar pattern was observed when We identified gene family expansions for proteases, chit-
comparing C. militaris, M. anisopliae and the plant inases, lipases and protein kinases in C. militaris when
pathogen Fusarium graminearum (Figure 3b). Compara- compared with phytopathogenic fungi, whereas gene
tive genomic analysis of the three insect pathogens family contractions occurred for glycoside hydrolases
found that the percentage of species-specific genes is (GHs; P = 0.0144; FDR = 0.02), cutinases (P = 0.0065;
much higher in C. militaris (13.7%) compared to M. FDR = 0.0226) and pectin lyases (P = 0.0245; FDR =
anisopliae (4.8%) and M. acridum (3.5%) (Figure 2b). 0.0284) (Table S1 in Additional file 1). The largest
Based on the identities between orthologous proteins, C. family expansions were for proteases. The C. militaris
militaris displays an average of approximately 63% genome contains 61 families of proteases but most of
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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.
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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.
them were included in families of serine proteases (180/ bacterial-like chymotrypsin identified in M. anisopliae
381) and metallopeptidases (108/381) (Table S5 in Addi- [21] is absent in M. acridum [11] but present as two
tional file 1). Gene expansions within the subtilisin (P = copies in C. militaris. The A01 aspartyl proteases are
0.0109; FDR = 0.0189) and trypsin (P = 0.0077; FDR = virulence factors of both mammalian and plant patho-
0.0178) families are consistent with their being virulence gens because of their ability to cleave an array of host
factors in insect pathogens [11]. However, different proteins [22]. Compared to phytopathogenic fungi (aver-
families of proteases are expanded in Metarhizium spp. age 17), their number is significantly (P = 0.0059; FDR =
and C. militaris, consistent with each lineage ?reinvent- 0.0057) expanded in the three insect pathogens (average
ing the wheel? during the evolution of entomopathogeni- 24) (Table S4 in Additional file 1).
city. Thus, relative to Metarhizium spp., the S01 trypsin Compared to many plant pathogens, Metarhizium spp.
and S08 subtilisin subfamilies are smaller and the S53 and C. militaris have fewer cutinases for degrading plant
subfamily is larger (Table S6 in Additional file 1). The cell walls (Table S1 in Additional file 1). They also have
C. militaris genome has 12 trypsin genes compared to 4 fewer (average 137, P < 0.05) GHs than plant pathogens
or less in plant pathogens. It lacks four subfamilies of (average 199), including the lack of 20 GH families used
trypsins present in M. anisopliae. Interestingly, the by most plant pathogens and saprobes to target plant
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Table 2 Genome-wide analysis of C. militaris gene sets
Characteristics C. militaris CMM corea CMA restrictedb CMC restrictedc CCM specific
Number of genes 9,684 7,981 217 158 1,328
Mean gene length (bp) 1,742 1,885 1,445 1,440 967
Mean number of introns per gene 1.99 2.05 1.77 1.69 1.71
Percentage genes without introns 21.3 20.1 22.6 28.5 27.5
Percentage GC content (excluding introns) 58.6 58.6 70.7 58.7 58.3
Number of InterproScan protein families 2,644 2,552 69 52 112
Number of secreted proteins 1,572 1,250 45 13 264
Number of PHI genesd 1,547 1,539 4 2 2
Number of TSA proteases 68 65 3 0 0
Number of MFS genes 245 242 0 2 1
Number of cytochrome P450s 57 56 0 1 0
Number of Pth11-like GPCRs 18 18 0 0 0
Number of protein kinases 167 167 0 0 0
Number of transcription factors 123 120 2 1 0
Number of glycoside hydrolases 105 103 2 0 0
Number of SM backbone genes 28 28 0 0 0
Number of horizontally transferred genes 49 30 5 1 12
Number of orthologs in M. anisopliae 6,863 6,705 158 NA NA
Number of orthologs in M. acridum 6,762 6,644 NA 118 NA
Number of orthologs in F. graminearum 6,740 6,376 106 89 169
Number of orthologs in M. oryzae 6,219 5,937 90 80 112
Percentage identity to M. anisopliae orthologs 63.4 63.7 51.3 NA NA
Percentage identity to M. acridum orthologs 63.4 63.6 NA 51.2 NA
Percentage identity to F. graminearum orthologs 61.6 62.3 51.9 52.8 46.7
Percentage identity to M. oryzae orthologs 56.0 56.4 48.3 48.0 45.3
aCMM core: C. militaris (CCM), M. anisopliae and M. acridum genes grouped with a cutoff E value of 1e-5 during reciprocal Blast analysis. bCMA restricted: C.
militias and M. anisopliae restricted genes grouped with a cutoff E value of 1e-5. cCMC restricted: C. militaris and M. acridum restricted genes grouped at a cutoff
E value of 1e-5. dPHI genes, pathogen-host interaction genes predicted by blast analysis against the PHI database [72]. Identity was estimated at the amino acid
level. GPCR, G-protein coupled receptor; MFS, major facilitator superfamily; NA, not available; SM, secondary metabolite; TSA, trypsin, subtilisin and aspartyl
protease.
cell walls - for example, GH6, GH7 and GH61 cellu- xenobiotics and the biosynthesis of secondary metabo-
lases, GH10 and GH11 xylanases, GH28 pectinases and lites [25]. C. militaris has only about half as many CYPs
GH78 rhamnosidases (Table S7 in Additional file 1). as Metarhizium spp., and most other fungi (Table S8 in
There are also significant differences in the spectrum of Additional file 1). Seventy CYP subfamilies present in
enzymes produced by the entomopathogens. For exam- M. anisopliae and/or M. acridum are absent in C. mili-
ple, compared to M. anisopliae, C. militaris has few taris. Of particular interest, C. militaris lacks CYP55,
xyloglucosyl transferases (GH16) for xyloglucan catabo- CYP58 and CYP65. CYP55 is a nitric oxide reductase
lism and lacks a-glucuronidases (GH115) active on required for denitrification [25]. Thus, unlike most fila-
xylan oligomers or polymeric xylan [23]. Consistent with mentous fungi, C. militaris may not respond to hypoxia
this, C. militaris grows very poorly on xylose when com- through the bacterial ammonia fermentation mechan-
pared with M. anisopliae (Figure S2 in Additional file 2). ism. The absence of CYP58 (trichodiene oxygenase) and
A phosphoketolase MPK1 involved in pentose metabo- CYP65 (trichothecene C-15 hydroxylase) suggests that
lism is required for full virulence of M. anisopliae [24], C. militaris will not produce the mycotoxin trichothe-
but the homolog is absent in C. militaris. However, cene [26]. M. anisopliae can efficiently metabolize insect
GH18 chitinases similar to those used by Metarhizium epicuticle alkanes [27]. The CYP52 subfamily for alkane
to degrade insect cuticles [11] are well represented in hydroxylation [25] is well represented in Cordyceps.
the C. militaris genome (20 in CCM versus 30 in MAA The major facilitator superfamily (MFS) and ATP-
and 19 in MAC) relative to plant pathogens (average 11) binding cassette (ABC) transporters are the two biggest
(Table S7 in Additional file 1). families of fungal transporters. Members of the former
Cytochrome P450s (CYPs) play essential roles in fun- typically function as nutrient symporters and drug anti-
gal physiologies, including detoxification, degradation of porters, whereas the latter are more often implicated in
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defense against toxic metabolites [28]. C. militaris has Strain Cm01 forms fruiting bodies on caterpillar
approximately half (123) the number of these transpor- pupae that lack perithecia and ascospores (Figure 5a-e).
ters as Metarhizium (269 in MAA and 236 in MAC) Thus, it is the first ascomycete species reported to fruit
(Table S9 in Additional file 1). The MFS transporters without an opposite mating-type partner. Other C. mili-
that are underrepresented in Cordyceps include the car- taris isolates could also fruit sterilely with a single mat-
bohydrate symporters (37 in CCM versus 48 in MAA, ing-type locus (Figure 6a, b). However, a hybrid strain,
51 in MAC and an average of 58 in plant pathogens), Cm06, with both MAT1-1 and MAT1-2 loci produced
vitamin B2 (riboflavin) transporters (2 in CCM versus sexual perithecia and ascospores (Figure 5f, g). In addi-
17 each in Metarhizium species and an average of 4 in tion, the sexual structures could be similarly re-formed
plant pathogens) and multidrug antiporters (23 in CCM after inoculation of the caterpillar pupae with different
versus 110 in MAA, 77 in MAC and an average of 10 in ratios of MAT1-1 and MAT1-2 isolate conidia (Figure
plant pathogens). Consistent with their having many 6c-e), confirming that C. militaris is heterothallic. PCR
multidrug transporters, Metarhizium spp. are resistant examination of 18 field-collected strains identified three
to diverse antibiotics and fungicides [29]. Cordyceps has containing both MAT1-1 and MAT1-2 loci (Figure 5h).
more ABC-type drug and metal resistant proteins than However, 28 out of 30 single spore isolates of the Cm06
Metarhizium and plant pathogens (63 in CCM, 56 in strain belonged to the MAT1-1 mating-type (Figure 5i).
MAA, 51 in MAC and an average of 54 in plant patho- A similar unequal prevalence of mating types occurs in
gens). The amino acid and dipeptide transporters are the dermatophyte fungus [31].
similarly represented in the three insect pathogens and
other fungi (46 in CCM versus 53 in MAA, 49 in MAC Metabolism of medically active components and
and an average of 45 in plant pathogens). mycotoxins
Fungal G-protein coupled receptors (GPCRs) are One of the main pharmaceutically active components of
required for pheromone/nutrient sensing and host C. militaris is cordycepin [5,6], which is structurally
recognition [11]. Thus, the Pth11-like GPCR of Magna- similar to 2?-deoxyadenosine (Figure 7a). C. militaris
porthe mediates cell differentiation in responses to plant possesses most of the genes required for metabolism of
inductive cues [30]. C. militaris has fewer GPCRs than adenine and adenosine except for lacking a ribonucleo-
Metarhizium spp. and is particularly impoverished in tide trisphosphate reductase (RNR; converts ATP to
Pth11-like GPCRs (Table S10 in Additional file 1). C. dATP) and a deoxyadenosine kinase (converts deoxyade-
militaris has a similar number (167) of protein kinases nosine to dAMP) (Figure 7b; Table S13 in Additional
as M. anisopliae (161) but less than M. acridum (192) file 1). It has been suggested that the biosynthesis of
(Table S11 in Additional file 1). Like other fungi, fungal cordycepin proceeds through a reductive mechanism as
specific transcription factors (TFs) and zinc finger TFs described for the formation of 2?-deoxyadenosine [32].
represent the two largest classes of TFs in C. militaris However, C. militaris resembles Metarhizium and other
and their numbers are similar to those of other fungi cordycepin non-producing fungi in having only two
(Table S1 in Additional file 1). highly conserved subunits of class I RNRs (Figure S3 in
Additional file 2). The substrates for class I RNRs are
Mating-type and sexuality analysis ADP, GDP, CDP and UDP but not TDP or nucleosides,
The fruiting bodies of Cordyceps spp. are the most com- and as the reductive reaction proceeds via a free radical
monly sold traditional Chinese medicine products [5]. mechanism [33], C. militaris RNRs will not be involved
However, the sexual cycle and fruiting of C. militaris is in cordycepin production.
poorly understood. We only identified a MAT1-1 mat- Contamination of food and feed by mycotoxins is a
ing-type locus, including MAT1-1-1 and MAT1-1-2 longstanding threat to the health of humans and animals
genes, in the sequenced Cm01 strain, suggesting that C. [26]. C. militaris has been consumed for hundreds of
militaris is heterothallic (Figure 4a). A single mating- years, implying safety, but the genome data allowed us
type locus was also found in M. anisopliae (MAT1-1) to make the first comprehensive inventory of Cordyceps
and M. acridum (MAT1-2). Like aspergilli [10], the idio- genes involved in biosynthesis of secondary metabolites
morphic regions of the three insect pathogens are highly for comparison with known mycotoxins. There are
divergent (Figure 4a). The MAT1-1 locus of M. aniso- fewer secondary metabolite core genes in C. militaris
pliae contains a MAT1-1-3 gene but lacks the MAT1-1- relative to Metarhizium spp. or plant pathogens (Table
2 gene present in C. militaris. Except for the mating- 3). In comparison to Metarhizium spp., Cordyceps has
type locus region, most A. nidulans and N. crassa genes fewer terpenoid synthases, polyketide synthases (PKSs)
involved in mating, fruiting, karyogamy and meiosis are and non-ribosomal peptide synthetases (NRPSs). Phylo-
also present in insect pathogens (Table S12 in Addi- genetic analysis of Cordyceps PKS and PKS-like genes
tional file 1). using the ketoacyl CoA synthase (KS) domain sequences
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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).
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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.
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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.
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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.
found that the C. militaris proteins grouped into differ- different from mycotoxin PKSs (Figure 8b). The further
ent clusters compared to PKSs for known mycotoxins survey showed that the CCM_00603 protein has only
(Figure 8a). In addition, modular analysis indicated that, 27% identity with PatK and the gene cluster for patulin
except for CCM_00603, which has a similar domain biosynthesis is absent in C. militaris (Table S14 in Addi-
organization to the Aspergillus clavatus PatK gene for tional file 1). This suggests that C. militaris PKSs do not
patulin biosynthesis, C. militaris PKSs are structurally produce patulin or other known human mycotoxins.
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Table 3 Numbers of core genes involved in the of undifferentiated mycelia from Sabouraud dextrose
biosynthesis of secondary metabolites in different fungi broth (SDB) culture with developmental stages on cater-
Core gene CCM MAA MAC FG MO BC SS NC AN pillar pupae defined as nascent (14 days, termed as sam-
DMAT 1 5 3 0 3 1 1 1 6 ple FB1), stalk formation (29 days, FB2) and mature
TC 3 3 3 3 3 3 3 3 5 fruiting bodies (59 days, FB3) (Figure 5a-c). Of the 9,684
TS 2 8 6 11 8 7 1 2 5 genes, more than 63% were expressed during both
FAS 1 2 2 1 1 1 1 1 1 undifferentiated hyphal growth and formation of fruiting
GGPS 3 4 4 3 3 0 0 1 0 bodies (Table S16 in Additional file 1). Relative to the
NRPS 5 14 13 10 5 6 5 3 11 growth in SDB, more than 900 genes were significantly
NRPS-like 8 9 8 11 6 8 5 3 12 (P < 0.05; FDR < 0.001) up-regulated while around
PKS 9 24 13 14 12 16 16 6 24 2,000 genes were down-regulated during fungal fruiting
PKS-like 2 3 4 1 3 6 2 2 4 (Figure 9a). A Pearson correlation analysis indicated that
HYBRID 3 5 1 1 3 0 0 0 1 transcriptional profiles at the different stages of fruiting
Total 37 77 57 55 47 48 34 22 69 body formation more closely resembled each other than
Core genes encoding: DMAT, dimethylallyl tryptophan synthase; TC, terpenoid they resembled the transcriptomes of undifferentiated
cyclase; TS, terpenoid synthase; FAS, fatty-acid synthase; GGPS, geranylgeranyl mycelia (Figure S6a in Additional file 2). This is consis-
diphosphate synthase; NRPS, non-ribosomal peptide synthetase; PKS, tent with a Venn diagram analysis of the commonest
polyketide synthetase; HYBRID, hybrid PKS-NRPS enzyme. Fungal species:
CCM, C. militaris; MAA, M. anisopliae; MAC, M. acridum; FG, F. graminearum; co-expressed genes between different samples (Figure
MO, M. oryzae; BC, Botrytis cinerea; SS, Sclerotinia sclerotiorum; NC, N. crassa; S6b in Additional file 2). Of the 100 most highly
AN, A. nidulans.
expressed genes in developing C. militaris fruiting
bodies, 26 (FB1), 31 (FB2) and 37 (FB3) are functionally
Similarly, phylogenetic and modular analyses indicated uncharacterized (Table S17 in Additional file 1). This
that Cordyceps NRPSs had different protein structures suggests that the genes with unknown function are
than any NRPSs involved in production of known myco- more likely to be stringently regulated and involved in
toxins like enniatin, HC-toxin and gliotoxin (Figure S4 developmental processes than orthologs of genes with
in Additional file 2). known function. These genes are thereby the targets for
The mycotoxin ergot alkaloids have a wide range of future functional studies. In general, the genes involved
biological activities and are important in pharmaceuti- in cell wall structure and biogenesis, detoxification, pro-
cals and agriculture [26]. Dimethylallyl tryptophan tein degradation and amino acid transportation were
synthase (DMAT) catalyzes the alkylation of L-trypto- significantly up-regulated during formation of fruiting
phan, the first committed step in the ergot alkaloid bio- structures. In contrast, most of the genes specifically up-
synthetic pathway [34]. C. militaris has one putative regulated by undifferentiated SDB cultures were
DMAT gene (CCM_04410), in contrast to five in M. involved in rapid growth and carbohydrate metabolism.
anisopliae and three in M. acridum (Table 3). A phylo- Concomitant with fruiting structure maturation, the
genetic analysis showed that CCM_04410 is not clus- genes for cytoskeletal organization, cell cycle and sec-
tered with the Claviceps DMAT clade involved in ergot ondary metabolism were up-regulated.
alkaloid production (Figure S5 in Additional file 2). The Unlike other fungi, C. militaris can fruit sterilely in
trichothecenes T-2 toxin and deoxynivalenol (type B tri- the absence of a sexual partner (Figure 5a-c). Perhaps
chothecene) are natural fungal products that are toxic to because of this, 31 of the 42 C. militaris orthologs of
both animals and plants [35]. Consistent with lacking sex-related genes identified in other ascomycetes were
CYP58 and CYP65, the C. militaris genome also lacks not expressed or transcribed at low levels (< 10 tran-
trichodiene synthase (Table S15 in Additional file 1). scripts per million tags (TPM)) in sterile fruiting bodies
Thus, unlike Fusarium [26], C. militaris is not predicted (Table S18 in Additional file 1). However, in some
to produce trichothecene mycotoxins. The presence of cases, C. militaris expresses paralogous genes to those
terpenoid cyclase, terpenoid synthase, fatty-acid synthase employed by other fungi, suggesting they have co-opted
and geranylgeranyl diphosphate synthase genes in the C. different components of the same signal transduction
militaris genome suggests that the fungus is capable of pathways to fulfill similar functions. For example,
producing an array of metabolites, but the identity of GATA-type TFs are important for fruiting in both A.
these and their biological activities remain to be nidulans and N. crassa [36], but C. militaris fruiting
determined. structures expressed orthologs of these genes at very
low levels or not at all (Table S19 in Additional file 1).
Transcriptional regulation of fruiting body development In contrast, the Zn2Cys6-type TFs were highly tran-
To identify genes associated with C. militaris fruiting scribed during fruiting but not in undifferentiated fungal
body development, we compared the expression profiles mycelia - for example, CCM_01809 and CCM_09644
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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 9b) - indicating that Zn2Cys6 type TFs are pre- orthologous genes were not transcribed (CCM_04200
dominately involved in the major developmental switch versus AN1017) or transcribed at low levels
of production of fruiting structures. (CCM_01235 versus NCU02393) by C. militaris (Table
Pheromone receptors, that is, GPCRs, control fungal S19 in Additional file 1). However, Cordyceps sharply
fruiting body formation and sexual cycle but not vegeta- up-regulated (P < 0.05, FDR < 0.001) a MAPK paralog
tive growth [36]. The pheromone receptor of C. mili- (CCM_09637) as well as a calcium regulated kinase
taris has not been identified. In comparison to (CaMK, CCM_06085) (Figure 9c). These data, taken in
undifferentiated mycelial growth, a putative pheromone conjunction with the single adenylate cyclase
receptor (CCM_01499) and a Pth11-like GPCR (CCM_06928) not being transcribed and the low level
(CCM_03015) were significantly up-regulated (P < 0.05, expression of both protein kinase A (PKA; CCM_03352)
FDR < 0.001), respectively, during initiation of fruiting and Rap GTPase (CCM_01391), indicate that fruiting by
body formation. Mitogen-activated protein kinase C. militaris in the absence of a partner is more depen-
(MAPK) genes are required for fruiting in Aspergillus dent on the MAPK pathway than the cAMP-dependent
(AN1017) and Neurospora (NC02393) [36], but PKA pathway (Figure 10).
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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.
Discussion involved in interactions with insect hosts. Cordyceps
We report here the first genome analysis of a Cordyceps resembles Metarhizium spp. in having a very high per-
species, the medicinal lepidopteran pathogen C. mili- centage of secreted proteins relative to plant pathogens
taris, and show that the fungus is capable of fruiting and saprophytes and expanded families of proteases and
without an opposite mating-type partner. We also show chitinases with targets in insect hosts. However, insect-
that it lacks genes known to be involved in production killing strategies may differ between Cordyceps and
of human mycotoxins. Being an insect pathogen, the C. Metarhizium due to differences in gene content. Mat-
militaris genome contains thousands of genes putatively ing-type analysis indicated that sexual reproduction in
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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.
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C. militaris is heterothallic. Transcriptional profiling lipid storage and appressorium penetration [41], and an
indicated that fruiting of the MAT1-1 C. militaris strain osmosensor (CCM_04885 versus MAA_01551) to med-
involves induction of the MAPK pathway, but unlike iate adaptation to the insect hemocoel [42]. Homologs
other homothallic or heterothallic fungi, the PKA path- of these genes are broadly distributed among ascomy-
way was not up-regulated. It remains to be determined cetes, indicative of an ancient origin. However, Cordy-
whether this reflects the very unusual ability of C. mili- ceps lacks other Metarhizium pathogenicity-related
taris to produce fruiting bodies without an opposite genes, including a collagen-like protein to evade the
mating-type partner. host immune system [43], a phosphoketolase for pentose
Aside from knowing that C. militaris infects lepidop- metabolism [24] and the adhesins to mediate spore
teran pupae [37], the life cycle of C. militaris in nature adhesions to insect and plant surfaces [44]. The absence
is poorly understood [8]. Following disease, survival in of key components of the Metarhizium entomopatho-
soil may depend on the sexual stage of Cordyceps pro- genicity ?toolkit? from C. militaris indicates that it has
viding resilient long-lived ascospores as described in evolved different determinants to mediate its interac-
other fungi [38]. Micro-cycle conidiation from germi- tions with insects.
nated ascospores could adapt the fungus to nutrient Like N. crassa and F. graminearum, C. miltaris lacks
poor niches (Figure 1). Metarhizium does not produce highly similar paralogs, a hallmark of the repeat-induced
ascospores but flourishes in plant rhizospheres, which point mutation (RIP) mechanism [20]. C. militaris has
thus provide an alternative habitat in the absence of an ortholog (CCM_03609) of the N. crassa RIP defective
insect hosts. C. militaris can grow on germinated soy- gene (NCU02034), a cytosine methyltranserase essential
beans [39], suggesting a potential for an association with for RIP [45]. The high C?T and G?A mutation bias in
plants. However, relative to Metarhizium and most the C. militaris genome and the readiness of C. miltaris
other ascomycetes, many protein families are smaller in to undergo the sexual cycle suggests that RIP is com-
the C. militaris genome, especially serine proteases, monplace in C. militaris like many ascomycetes
GHs, CYPs, MFS transporters and signal transduction [10,15,19]. Since RIP can function effectively against
factors. These families would be involved in scavenging selfish DNAs [46], it likely contributes, at least in part,
for nutrients, avoidance of host defenses and toxins and to C. militaris having few DNA type transposon
other processes related to pathogenicity and a saprobic encoded genes, that is, transposases [15].
lifestyle. Around two-thirds of these protein families There are many more orphan genes in Cordyceps than
include pathogen-host interaction genes in plant-asso- in Metarhizium spp., underscoring that much about the
ciated fungi (Table S4 in Additional file 1). Further stu- proteome of Cordyceps spp. remains unknown. It is
dies on the ecology of Cordyceps spp. will shed more speculated that orphan genes arise from gene duplica-
light on the relevance of the C. militaris genome to the tion, shuffling of gene fragments, mobile element
evolution of gene families in relation to acquisition/loss effects, mutation of existing sequences, horizontal gene
of capability for dual plant/insect colonization and host transfer and de novo origination from non-coding
range specialization. DNAs [47]. De novo creation of new genes is probably
The phylogenomic analysis demonstrated that the rare [48]. A role for mobile element effects is also unli-
lineage leading to Cordyceps spp. diverged after most kely given how few putative transposase genes are pre-
well known plant pathogens, including F. graminearum, sent in the C. militaris genome. Putative horizontal
but 130 MYA before Metarhizium diverged from the gene transfer genes are even fewer in Cordyceps than in
grass endophyte Epichlo? festucae. The estimate of a Metarhizium. Thus, the numerous orphans in the C.
Triassic-Jurassic boundary origin for the Cordyceps line- militaris genome most likely arose from frequent muta-
age and the post-Cretaceous origin of Metarhizium spp. tions caused by RIP in existing (duplicated) sequences.
is consistent with the hypocrealean fungi of Cordycipita- Just as the Metarhizium-specific collagen-like protein is
ceae (includes Cordyceps spp.), Clavicipitaceae (includes essentially required to camouflage cells from host
Metarhizium spp.) and Ophiocordycipitaceae splitting immune recognition [43]. The transcriptome data
about the same time as insects and angiosperms were showed that 428 of the 1,329 orphan genes were tran-
diversifying [40]. Families of proteases and chitinases are scribed during fruiting. Of the 100 most highly
not expanded or lost in the E. festucae genome as they expressed genes in developing C. militaris fruiting
are in Cordyceps and Metarhizium, exemplifying conver- bodies, about one-third are orphans (Table S17 in Addi-
gent evolution to insect pathogenicity. Besides proteases tional file 1), underscoring the potential of orphans to
and chitinases, experimentally verified Metarhizium have specific functions. Likewise, genes that are appar-
virulence-associated genes with homologs in the C. mili- ently unique to the mushroom Schizophyllum commune
taris genome include a perilipin-like protein are more likely to be expressed during mushroom for-
(CCM_06103 versus MAA_08819) to control cellular mation [49].
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Concern has been raised about the possibility of produces medicinal compounds and so further their
harmful side effects of traditional Chinese medicines, exploitation.
including Cordyceps [50]. Consistent with genotoxicity
and cytotoxicity assays that show Cordyceps products to Materials and methods
be safe for consumption [51], there is no evidence in Fungal strains
the C. militaris genome for genes involved in the pro- C. militaris strain Cm01 (CGMCC 3.14242) was selected
duction of known mycotoxins. However, safety could for genome sequencing as it is culturally stable and
only be completely verified by meticulous profiling of commercialized in China. The culture was maintained
the metabolites produced by the fungus under diverse either on artificial medium or silkworm pupae as pre-
growth conditions. The C. militaris genome data will viously described [12]. Several different C. militaris
facilitate these processes as well as help with elucidation strains were included in this study for PCR genotyping
of the biosynthetic pathways of different metabolites. of mating-type genes (Figure 5h).
The analysis of C. militaris genome indicates that it is
sexually heterothallic, but strikingly, both the MAT1-1 Genome sequencing and assembly
single mating-type and MAT1-1/MAT1-2 hybrid strains The genome of C. militaris strain Cm01 was shotgun
can form fruiting bodies, which means that C. militaris sequenced using a Roche 454 GS FLX system for mas-
is capable of fruiting without a partner. Single mating- sively parallel pyrosequencing for 2.25 runs at the Chi-
type (haploid) fruiting has also been observed in the nese National Human Genome Center (Shanghai,
human pathogens Cryptococcus neoformans and Can- China). This resulted in 951 Mb of sequence data (29.6
dida albicans [52]. Given that perithecia and ascospores ? coverage) with an average read length of 385 bp.
are not produced by MAT1-1 fruiting bodies, C. mili- Assembly was performed using the Newbler software
taris haploid fruiting is different from the same-sex mat- (v2.3) within the Roche 454 suite package [56], which
ing and fruiting of C. neoformans, in which produced 597 contigs with a total size of 32.2 Mb. For
diploidization and meiosis can occur. In budding yeast, sequence scaffolding, a DNA library of 2- to 5-kb inserts
meiotic recombination is initiated by the formation of was generated and sequenced with an ABI SOLiD sys-
double-strand breaks catalyzed by SPO11, a meiosis-spe- tem (Carlsbad, California, USA). This resulted in 3.8 Gb
cific endonuclease [53]. The meiosis-specific recombi- of mate-pair reads (117.4 ? coverage) to improve
nase DMC1 and the DNA repair enzyme RAD51 then sequence quality and construct scaffolds. By mapping
co-localize to double-strand breaks and function the reads to contigs, 578 contigs were assembled into 13
together for meiotic recombination [54]. The C. mili- scaffolds and 19 contigs less than 2 kb left outside. The
taris homologue of yeast SPO11 (CCM_09527) was up- raw data of 454 and SOLiD reads have been deposited
regulated more than five-fold during fruiting body at NCBI?s Sequence Read Archive under accession num-
maturation (the TPM ratio of FB2/FB1 = 8.1; FB3/FB2 = ber SRA047932 and the whole project has been depos-
5.7). Intriguingly, the C. militaris genome lacks a yeast ited at DDBJ/EMBL/GenBank under accession number
RAD51 ortholog, but its DMC1 ortholog (CCM_06822) AEVU00000000.
contains a RAD51 domain. CCM_06822 was not
expressed by C. militaris during fruiting, which may Annotation
explain why the C. militaris MAT1-1 strain forms fruit- To maximize gene prediction accuracy, the gene struc-
ing bodies without meiosis. Consistent with this, a puta- tures of Cordyceps were predicted with a combination of
tive cyclin dependent kinase 7 (CDK7; CCM_03900) was different algorithms plus manual inspections [11,57].
up-regulated during fungal fruiting (Table S19 in Addi- The inconsistent open reading frames were individually
tional file 1). Orthologs of CDK7 initiate DNA synthesis subject to Blast searches against the NCBI curated
and facilitate mitosis instead of meiosis [55]. refseq_protein database. The prediction with the best hit
was selected. Pseudogene identification was conducted
Conclusions with the pipeline of PseudoPipe with default settings
In conclusion, we report on the genome sequencing, [58]. The potential secreted proteins of C. militaris and
comparative genome analysis and transcriptional regula- other fungal species included for comparison were pre-
tion of fruiting body development in the medicinal fun- dicted by SignaIP 3.0 analysis using a hidden Markov
gus C. militaris. The sequence data should markedly model [59]. Genome repetitive elements were analyzed
enhance the pace of molecular research on Cordyceps by Blast against the RepeatMasker library (Open 3.2.9)
biology, fungal sex and pathogenicity, and will have [60] and with the Tandem Repeats Finder [61]. The
impacts on the commercial production of fruiting struc- transposases/retrotransposases were classified by Blastp
tures. The genomic sequence will also be an essential analysis against the Repbase [62] plus manual
tool to unravel the mechanisms by which C. militaris inspections.
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Blast score ratio test compare the differences in protein family sizes between
BSR tests [14] were conducted to compare the differ- insect and plant pathogens. Estimation of FDR of P-
ences between C. militaris and the sequenced Metarhi- values was conducted using the program mafdr (Matlab
zum genomes and the plant pathogen F. graminearum, 7.8.0.347(R2009a)).
respectively. The BSR index for each reference protein is
calculated by dividing the query bit score by the refer- Analysis of genes involved in purine synthesis and
ence score and normalized from 0 to 1. A score of 1 secondary metabolism
indicates a perfect match while a score of 0 indicates no To model the biosynthesis of cordycepin, the purine
Blast match of a query protein in the reference pro- metabolic pathway in C. militaris was constructed based
teome. The normalized pairs of BSR indices were then on the KEGG (Kyoto Encyclopedia of Genes and Gen-
plotted using the Matlab (v7.0) program (Natick, Massa- omes) annotations [73]. To identify NRPS, PKS or
chusetts, USA). The same analysis was conducted for NRPS-PKS hybrid genes and gene clusters, the whole
the three genomes of C. militaris, M. anisopliae and F. genome data set was subjected to analysis with the pro-
graminearum. gram SMURF with default settings [74]. Modulation
analysis and domain extraction of different NRPS or
Orthology and phylogenomic analysis PKS proteins were conducted by Blast searching against
In total, 2,106 orthologous proteins were acquired by a the SBSPKS database [75]. For phylogenetic analysis, the
reciprocal Blast method with a cutoff E value of 1e-20 domain sequences were aligned with Clustal X 2.0 and
and a Blast alignment length greater than 60% of the the tree was generated using a Poisson model with
query sequence. Corresponding orthologous gene pro- 1,000 bootstrap replications and pair-wise deletions for
tein sequences were aligned with Clustal X 2.0 and the gaps or missing data. The mycotoxin-encoding PKSs
concatenated amino acid sequences were used for the used in the analysis include Gibberella zeae PKS4
generation of a maximum likelihood phylogenomic tree (ABB90283) and PKS13 (ABB90282) for the biosynthesis
with the program TREE-PUZZLE [63] using a Dayhoff of zearalenones [76], Aspergillus ochraceus PKS
model. The divergence time between species was esti- (AAT92023) for ochratoxin [77], A. clavatus PatK
mated with the program r8s using a Langley-Fitch (ACLA_093660) for patulin [78], Gibberella moniliformis
model [64] by calibration with the origin of the Ascomy- Fum1p (AAD43562) for fumonisin [79], Monascus pur-
cota at 500 to 650 MYA [65]. pureus PKS (BAD44749) for citrinin [80], Aspergillus fla-
vus PksA (AAS90093) for aflatoxin [81] and A. nidulans
Protein family classifications StcA (Q12397) for sterigmatocystin [82]. The myco-
Whole genome protein families were classified by Inter- toxin-encoding NRPSs included in the analysis are A.
proScan [66] and Pfam [67] analysis. The families of fumigatus Glip (AAW03307) for gliotoxin [83], Fusar-
proteases were identified by Blastp searching against the ium equiseti NRPS (CAA79245) for enniatin [84],
MEROPS peptidase database release 9.4 with a cutoff E Cochliobolus carbonum NRPS (AAA33023) for HC-
value of 1e-20 [68]. The CYPs were named according to toxin [85] and Tolypocladium inflatum NRPS
the classifications collected at the P450 database [69]. (CAA82227) for cyclosporin [86].
Transporters were classified based on the Transport
Classification Database [70]. Kinases were classified by Transcriptome analysis
Blastp analysis against the KinBase database with a cut- Conidia of C. militaris from day 14 potato dextrose agar
off E value of 1e-10 [68]. Carbohydrate-active enzymes were inoculated into SDB and the undifferentiated
were classified by local Blastp searching against a library mycelia harvested after a 72-hour incubation at 25?C,
of catalytic and carbohydrate-binding module enzymes 180 rpm. The transcriptome of the mycelia provided a
[68]. G-protein-coupled receptors were selected from control for comparison with the transcriptomes of fruit-
the best hits to GPCRDB sequences [71] and by confir- ing bodies. Chinese Tussah silkmoth (Antheraea pernyi)
mation that they contained seven transmembrane pupae were injected with 50 ?l of a conidial suspension
helices with the amino terminus outside and the car- (5 ? 106 conidia/ml) and incubated at 22?C in a 12:12
boxyl terminus inside the plasma membrane. Homologs hour light:dark cycle for up to 14 days to allow emer-
of the Magnaporthe Pth11-like GPCRs [30] were identi- gence of fruiting bodies (a stage designated as FB1), 29
fied by local Blastp analysis with a cutoff E value of 1e- days for half-grown fruiting bodies (FB2) and 59 days,
10. Putative Cordyceps virulence factors were identified by which time fruiting bodies were mature (FB3)
by searching against the pathogen-host interaction data- [11,12]. RNA was extracted with a Qiagen RNeasy kit
base [72] with a cutoff E value of 1e-5, plus additional plus on-column treatment with RNase-free DNase I
searches of known virulence genes reported in entomo- (Germantown, Maryland, USA). Messenger RNA was
pathogenic fungi. Two sample t-tests were conducted to purified and after reverse transcription into cDNA, the
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libraries were constructed for tag preparation according ribosomal peptide synthetase; PKA: protein kinase A; PKS: polyketide
to the massively parallel signature sequencing protocol synthase; RIP: repeat-induced point mutation; RNR: ribonucleotide
trisphosphate reductase; SDB: Sabouraud dextrose broth; TF: transcription
[87]. The tags were sequenced with an Illumina techni- factor; TPM: transcripts per million tags.
que. We omitted tags from further analysis if only one
copy was detected or it could be mapped to a different Acknowledgements
CSW was supported by the Ministry of Science and Technology of China
transcript. Other tags were mapped to the genome or (grant number 2009CB118904), the Ministry of Agriculture of China (grant
annotated genes if they possessed no more than one number 2009ZX08009-035B), the Science and Technology Commission of
nucleotide mismatch [11,88]. The abundance of each tag Shanghai Municipality (grant number 08DZ1970200) and the Chinese
Academy of Sciences (KSCX2-EW-N-06 and KSCX2-EW-G-16).
was converted to the value of transcripts per million
(TPM) for each mapped gene for expressional compari- Author details
1
son between samples. The significance of differential Key Laboratory of Insect Developmental and Evolutionary Biology, Institute
of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,
gene expression between samples and the FDR of P- Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
values were estimated for each individual gene with a 2Chinese National Human Genome Center at Shanghai, 250 Bibo Road,
Shanghai 201203, China. 3cutoff of P ? 0.05 and FDR ?0.001 [11,89]. The RNA_- Institute of Microbiology, Chinese Academy of
Sciences, 1 West Beichen Road, Beijing 100101, China. 4Department of
seq expression dataset is available at the NCBI?s Gene Entomology, University of Maryland, 4112 Plant Sciences Building, College
Expression Omnibus under the accession code Park, Maryland 20742, USA.
GSE28001.
Authors? contributions
CSW initiated and designed the study. PZ, YLX, GHX, CHX, YH and YZ
Additional material annotated genes and performed protein family analysis; GHX and HX
performed phylogenetic and transcriptome analysis; PZ and SWZ conducted
fruiting body induction, and DNA and RNA extraction; HJZ, SYW and GPZ
Additional file 1: Comparative genomics analysis of C. militaris. The performed genome sequencing and assembly; PZ and GHX performed
file contains additional information on genomic properties and transcriptome analysis; CSW and RJSL wrote the paper. All authors read and
comparative gene family analysis of C. militaris with other fungi approved the final manuscript.
comprising 19 tables provided in separate excel sheets. Table S1
summarizes major protein family sizes of different fungal species. Table Received: 4 July 2011 Revised: 10 November 2011
S2 provides a comparison of transposase genes among three insect Accepted: 23 November 2011 Published: 23 November 2011
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