Insights from the genome of Ophiocordyceps polyrhachis-furcata to pathogenicity and host specificity in insect fungi

Abstract

Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants’ behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain insights into its biology and the emergence of host specificity.
O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum, Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes (proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2) retrotransposable elements.
The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved. The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.

Full text

R E S E A R C H A R T I C L E Open Access
Insights from the genome of Ophiocordyceps
polyrhachis-furcata to pathogenicity and
host specificity in insect fungi
Duangdao Wichadakul
1,2†
, Noppol Kobmoo
1†
, Supawadee Ingsriswang
1*
, Sithichoke Tangphatsornruang
1
,
Duriya Chantasingh
1
, Janet Jennifer Luangsa-ard
1*
and Lily Eurwilaichitr
1
Abstract
Background: Ophiocordyceps unilateralis is an outstanding insect fungus for its biology to manipulate host ants’
behavior and for its extreme host-specificity. Through the sequencing and annotation of Ophiocordyceps polyrhachis-
furcata, a species in the O. unilateralis species complex specific to the ant Polyrhachis furcata, comparative analyses on
genes involved in pathogenicity and virulence between this fungus and other fungi were undertaken in order to gain
insights into its biology and the emergence of host specificity.
Results: O. polyrhachis-furcata possesses various genes implicated in pathogenicity and virulence common with other
fungi. Overall, this fungus possesses protein-coding genes similar to those found on other insect fungi with available
genomic resources (Beauveria bassiana, Metarhizium robertsii (formerly classified as M. anisopliae s.l.), Metarhizium acridum,
Cordyceps militaris, Ophiocordyceps sinensis). Comparative analyses in regard of the host ranges of insect fungi showed a
tendency toward contractions of various gene families for narrow host-range species, including cuticle-degrading genes
(proteases, carbohydrate esterases) and some families of pathogen-host interaction (PHI) genes. For many families of
genes, O. polyrhachis-furcata had the least number of genes found; some genes commonly found in other insect fungi
are even absent (e.g. Class 1 hydrophobin). However, there are expansions of genes involved in 1) the production of
bacterial-like toxins in O. polyrhachis-furcata, compared with other entomopathogenic fungi, and 2)
retrotransposable elements.
Conclusions: The gain and loss of gene families helps us understand how fungal pathogenicity in insect hosts evolved.
The loss of various genes involved throughout the pathogenesis for O. unilateralis would result in a reduced capacity to
exploit larger ranges of hosts and therefore in the different level of host specificity, while the expansions of other gene
families suggest an adaptation to particular environments with unexpected strategies like oral toxicity, through the
production of bacterial-like toxins, or sophisticated mechanisms underlying pathogenicity through retrotransposons.
Keywords: Ophiocordyceps unilateralis, Genome, Next-generation sequencing, Comparative genomics, Host specificity,
Pathogenicity, Pathogen-host interaction
* Correspondence: supawadee@biotec.or.th;jajen@biotec.or.th
†
Equal contributors
1
National Center for Genetic Engineering and Biotechnology, National
Science and Technology Development Agency, 113 Thailand Science Park,
Phahonyothin Rd., Khlong Neung, Khlong Luang 12120 Pathum Thani,
Thailand
Full list of author information is available at the end of the article
© 2015 Wichadakul et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wichadakul et al. BMC Genomics (2015) 16:881
DOI 10.1186/s12864-015-2101-4
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Background
Fungi constitute one of the most diverse kingdoms of
living organisms including various forms and ecological
roles such as decomposers, mutualists or parasitical
symbionts. Their importance for industrial and agricul-
tural applications and their experimental tractability
made them useful and popular model for studying cell
biology and functional genomics in various biological
context. In this regard, the number of whole-genome se-
quence data from a wide variety of fungi has dramatic-
ally increased during the last decade [1].
Entomopathogenic fungi (or insect fungi) are also re-
ceiving growing attention with regard to the develop-
ment of genomic resources. On one hand, M. anisopliae
s.l. (including M. robertsii) and B. bassiana s.l., which
are widely used as agents for biological control have in-
stigated the development of genomic resources and ex-
perimental approaches in order to study virulence and
pathogenicity [2, 3]. On the other hand, other species
such as O. sinensis and C. militaris are widely used in
Asia as traditional Chinese medicine and has therefore
spurred interests for studying novel biosynthetic path-
way and sexual reproduction through genomic ap-
proaches [4, 5]. Insect fungi are also well known for the
production of interesting secondary metabolites such as
polyketides and non-ribosomal peptides whereas the
synthetic pathways have not yet been all elucidated [6].
In this study, we report a draft genome of O.
polyrachis-furcata, a species in the O. unilateralis spe-
cies complex, which is also a Hypocrealean entomo-
pathogenic fungus. This species is outstanding for its
ecological strategy as it modifies host ants’behavior in
order to favor its own dispersion (“death grip,”i.e. in-
fected ants climb into vegetation, bite vegetal mate-
rials then hang themselves upside down until death,
andconsideredasanextendedphenotypefromthe
fungus.) [7]. The biology of O. unilateralis still has
much to be discovered. The pathogenesis and molecu-
lar basis to pathogenicity are poorly known while the
molecular basis of behavior manipulation is a total
black box. The genome sequencing of this species will
provide a basis for further exploration on these issues.
Additionally, this fungus also differs from those previ-
ously cited regarding its host range. While M. anisopliae
(including M. robertsii)andB. bassiana have very broad
host ranges infecting several insect orders and could live
as plant endophytes and in the soil, O. sinensis,C. militaris
and O. unilateralis have narrower host ranges including
various lepidopteran families for C. militaris [8], a family
of Lepidoptera (Hepialidae) for O. sinensis [9], and only a
tribe (Camponotini) in a sub-family of ants (Formicinae)
for O. unilateralis [10]. Previous studies showed hidden
diversity of O. unilateralis associated to ant species, sug-
gesting a diversification through host specificity [10, 11].
A recent study suggested that O. unilateralis in Thailand
should be subdivided into three distinct species (O.
polyrhachis-furcata,O. camponoti-saundersi,O. camponoti-
leonardi) according to the host species [11]. In this study,
we focused on sequencing O. polyrhachis-furcata and ana-
lyzing its genes that were previously reported to be import-
ant to different steps of pathogenesis and thus virulence in
other insect fungi and fungal pathogens. Through compara-
tive genomics between our fungus and nineteen other fungi
including other entomopathogenic fungi (B. bassiana,M.
robertsii,M. acridum,C. militaris,O. sinensis), some fungal
plant pathogens (Magnaporthe oryzae,Botryotinia fuckeli-
ana,Fusarium graminearum,Sclerotinia sclerotiorum,Usti-
lago maydis,Verticillium alfalfae), opportunistic human
pathogenic fungi and yeast (Aspergillus fumigatus,Candida
albicans,Saccharomyces cerevisiae), a fungal pathogen of
amphibian (Batrachochytrium dendrobatidis)andsapro-
phytic fungi (Aspergillus nidulans,Neurospora crassa,
Coprinopsis cinerea)aswellasanon-pathogenicyeast
(Schizosaccharomyces pombe); we identified genes involved
in various steps of pathogenesis, investigated the common
attributes of being entomopathogenic and the extent to
which the host ranges have shaped their evolution as well
as enable the discovery of new biosynthetic pathways.
Results
General genome features
The genome of O. polyrhachis-furcata (strain BCC54312)
was sequenced in-house using the Roche 454 GS FLX sys-
tem. This resulted in 1.28 Gb of sequence data (37x cover-
age) with average read length of 652 bp. The shotgun
reads were de novo assembled resulting in 4419 contigs.
For sequence scaffolding, a DNA library of 3 kb, 6 kb, and
8 kb inserts were constructed and sent to Macrogen
(Seoul, Republic of Korea) for sequencing on Hiseq2000
platform (Illumina). 4419 contigs together with the mate-
pair libraries were assembled into 418 scaffolds (59 scaf-
folds > 1 kb; N50 3.3 Mb) with a total estimated genome
size of about 43 Mb. The assembled genome has been de-
posited as an NCBI’s Whole Genome Shotgun (WGS)
project under accession number LKCN00000000 and
the data of the sequenced samples deposited at the
NCBI’s BioSample database under the accession
number SAMN04099149.
While having an equivalent genome size to other in-
sect fungi (except O. sinensis),thegenomeofO.
polyrhachis-fucata BCC54312 was predicted to have
6793 protein coding genes. This number is close to that of
protein coding genes reported in [4] for O. sinensis and
substantially less than those reported for M. robertsii,M.
acridum,B. bassiana,andC. militaris which have broader
host ranges. Furthermore, the number of secreted proteins
as predicted by SignalP 4.0 [12] is less than half of those
four entomopathogenic genomes (Table 1).This number is
Wichadakul et al. BMC Genomics (2015) 16:881 Page 2 of 14
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reliable as the assembled genome was assessed to be as
complete as 96 % (4.5 % duplicated), 2.5 % fragmented,
and only 1.1 % missing. The set of annotated protein cod-
ing genes was assessed to be 95 % complete (9.2 % dupli-
cated), 2.8 % fragmented, and only 1.8 % missing.
Table 1 shows the overall genome features of O.
polyrhachis-furcata compared with other five entomo-
pathogenic fungi using the same versions of computer
programs and databases except for protein secretion.
A phylogenomic tree based on conserved proteins among
all taxa used in this study showed O. polyrhachis-furcata
to be closely related to O. sinensis which is from the same
family (Ophiocordycipitaceae) and clustered with other
fungi from the order Hypocreales including other entomo-
pathogenic fungi (Fig. 1).
Pathogen-Host Interaction genes
The potential virulence-associated genes in the O.
polyrhachis-furcata and nineteen other genomes were
identified by BLAST analysis against the pathogen-host
interaction (PHI) database [13]. This is a useful database
synthesizing a plethora of experimentally verified genes re-
lated to the mediation of fungal pathogens, including
Oomycetes, to cause disease and to provoke response
from the hosts (pathogenicity, virulence and effector
genes). We identified 1890 putative PHI genes in O.
polyrhachis-furcata, which is remarkably lower than in
other insect fungi with broader host ranges (see
Additional file 1: Table S1). The six species of ento-
mopathogenic fungi were clustered together according to
thenumberofspecificPHIgenes,showntoberelatedto
pathogenicity, which were identified across all the genomes
used for the analysis (Fig. 2). Particularly, O. polyrhachis-
furcata was the closest to O. sinensis. Also, the plant patho-
gens appeared close in terms of PHI genes abundance while
yeasts (S. cerevisiae, S. pombe, C. albicans), saprophytic
mushroom (C. cinerea) and the amphibian pathogen (B.
dendrobatidis) share similar patterns of PHI genes abun-
dance (Fig. 2 and Additional file 1: Table S1).
Although O. polyrhachis-furcata and O. sinensis have
generally less PHI genes compared with the other insect
fungi, some PHI gene families were specifically found or
in higher numbers in these two fungi. Interestingly, O.
polyrhachis-furcata contains more PHI:871 genes (8),
compared with other five entomopathogenic fungi (0 to
1). This class of genes was reported as virulence gene in
the rice blast fungus, M. grisea [14]. Among the twenty
genomes, all except O. polyrhachis-furcata and M. gri-
sea, contain at most two PHI:871 genes. Among the six
entomopathogenic fungi, PHI:113, PHI:1158, PHI:1276,
PHI:2314, PHI:449 and PHI:1278 genes were found only
in O. polyrhachis-furcata. PHI:113 was also reported in
M. grisea. Its disruption caused the loss of pathogenicity
against rice [15]. PHI:1158 was characterized in Myco-
sphaerella graminicola as resistance to azole fungicides
[16]. The gene deletion of PHI:2314 resulted in reduced
virulence of S. sclerotiorum and oxidative burst in adja-
cent uninfected cells of tomato (Nicotiana benthamiana)
[17]. The disruption of PHI:449 gene in Claviceps pur-
purea T5 reduced virulence against Rye [18] while the
disruption of PHI:1276, PHI:1278 in Gibberella zeae did
not affect pathogenicity [19]. O. polyrhachis-furcata and
O. sinensis contain PHI:292 [20], PHI:2198 [21],
PHI:2222 [22], PHI:2342 [23] and PHI:2534 [24] genes,
which are missing from the other four entomopatho-
genic fungi. The deletion of all of these genes except
PHI:2342 resulted in reduced-virulence phenotypes in
other fungal pathogens. For example, the lethality effect
on A. fumigatus Af293 comes from the double mutant of
PHI:2534 (the deletion of both ERG11A and ERG11B)
while each individual deletion can compensate the loss
of the other [24].
B. bassiana contain more copies of PHI:1139 (79),
PHI:820(23), PHI:821(19), PHI:823 (10), compared to
Table 1 Comparison of genome features among six entomopathogenic fungi
Features OPF OPSINE METANI METACR BEUBAS CORMIL
Size (Mb) 43 ~120 39 38.1 33.7 32.2
Coverage (fold) 37x(454) 241x 100x 107x 76.6x 147x
No.of scaffolds(>1 kb) 65 - 176 241 242 13
Scaffold N50 (Mb) 3.3 - ~2.0 0.33 0.73 4.55
%G + C content 45.2 46.1 51.5 50.0 51.5 51.4
%G + C in coding genes 59.7 - 54.4 54.1 56.6 58.6
Protein coding genes 6,799 6,972 10,582 9,849 10,366 9,684
Exons per gene 3.29 2.6 2.8 2.7 2.7 3
tRNAs 83/77 - 141 122 113 136
No. of secreted proteins 690 - 1,865 1,490 ~1886 1,572
No. of PHI genes 1,223 998 1,828 1,629 - 1,547
No. of Pth11-like GPCRs 14 - 54 40 23 18
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other five entomopathogenic fungi (5–15 for PHI:1139
and 1–2 for PHI:820, PHI:821, and PHI:823). Interestingly,
PHI:1139 in Xanthomonas oryzae is a plant avirulence de-
terminant [25]. The mutant phenotype of other three PHI
genes is resistant to chemical fungicides [26–28].
Another interesting PHI genes set consists of PHI:2240,
PHI:1555, PHI:812, and PHI:511 as O. polyrhachis-furcata
and O. sinensis have less number of these PHI genes com-
pared to other four entomopathogenic fungi. PHI:2240 was
reported as a plasma membrane-localized sucrose trans-
porter (Srt1); a fungal virulence factor, characterized from
corn smut fungus U. maydis [29]. PHI:1555 has not been
reported to affect pathogenicity while PHI:812 was reported
as a virulence gene in M. grisea [14]. Finally, PHI:511 was
reported as involving in drug sensitivity and virulence in C.
albicans [30] (see Additional file 2: Table S2).
Surface adhesion genes
The first step to cause disease is the attachment of the
infective propagules on the hosts’surface. This involves
hydrophobic interactions between the spore surface pro-
teins (e.g. adhesin, hydrophobin) and the lipid layer on
the epicuticle of insects. Hydrophobins can be classified
into two classes based on their hydropathy and solubility
characteristics [31]. In insect fungi, hydrophobins were pre-
viously shown to be involved in the formation of appresso-
rium in M. anisopliae [32] and B. bassiana [33, 34] as well
as virulence factors in M. brunneum [35]. Only Class 2
hydrophobins, which are represented by the orthologous
proteins MAA_01182 and MAC_09507 in M. robertsii and
M. acridum respectively, were identified in the genome of
O. polyrhachis-furcata. The genomes of the three other en-
tomopathogenic fungi also contain this class of hydropho-
bins. In addition, six other species (B. fuckeliana,F.
graminearum,M. oryzae,N. crassa,S. sclerotiorum and V.
alfalfae) have orthologous proteins of this class. Notably,
the protein BBA_03071 of B. bassiana,whichisannotated
as Hydrophobin-like protein at UniProt, was clustered to
the same orthologous group of the Class 2 hydrophobins of
the other entomopathogenic fungi while the annotated
Hydrophobin 2 of this species (BBA_00530: hyd2)could
not be clustered with the other species. The gene product
of hyd2 was reported as the major component of the B.
bassiana rodlet layer [33]. In contrast, no orthologous Class
1 hydrophobin was identified in O. polyrhachis-furcata.
The Class 1 hydrophobins MAA_10298 and MAC_04376,
reported in [2], and BBA_03015, reported in [3], were iden-
tified in C. militaris and O. sinensis as well as in eight other
Batrachochytrium dendrobatidis
Aspergillus fumigatus
Aspergillus nidulans
Verticillium alfalfae
Ophiocordyceps sinensis
Ophiocordyceps polyrhachis-furcat
a
Fusarium graminearum
Cordyceps militaris
Beauveria bassiana
Metarhizium robertsii
Metarhizium acridum
Neurospora crassa
Magnaporthe oryzae
Botryotinia fuckeliana
Sclerotinia sclerotiorum
Saccharomyces cerevisiae
Candida albicans
Schizosaccharomyces pombe
Ustilago maydis
Coprinosis cinerea
Fig. 1 A phylogenomic tree based on conserved proteins among taxa
Wichadakul et al. BMC Genomics (2015) 16:881 Page 4 of 14
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species, including A. fumigatus,A. nidulans, B. fuckeliana
(strain BcDW1), C. cinerea (strain Okayama-7/130/ATCC
MYA-4618/FGSC 9003), Gibberella zeae/F. graminearum
(anamorph), M. oryzae,S. sclerotiorum,U. maydis.
Beside hydrophobins, the MAD1 adhesin (MAA_03775),
characterized previously in M. robertsii, also provides adhe-
sion specifically to insect host surfaces, enhances the ex-
pression of genes related to i.e., germination, blastospore
formation, and thus affects virulence to caterpillars [36]. O.
polyrhachis-furcata as well as all other eighteen species
have multiple orthologous proteins of this adhesin. The
MAD2 adhesin (MAA_03807) allows adhesion of M.
robertsii to plant surfaces [36] but has no effects on fungal
differentiation and entomopathogenicity. Interestingly, only
six genomes, the V. alfalfae genome together with five
other from entomopathogenic fungi, excluding O. sinensis
have a single copy of MAD2 adhesin ortholog.
Insect cuticle degrading genes
Insect pathogens are expected to secrete large number of
enzymes for degrading insect cuticle. These include vari-
ous proteinases, particularly subtilisins, and other en-
zymes susceptible to degrade molecules present on the
insects’cuticles (i.e. chitinase and other glycoside hydro-
lases). O. polyrhachis-furcata and O. sinensis contain simi-
lar numbers of subtilisins, trypsins, and aspartyl proteases
which are much smaller than those of other four entomo-
pathogenic fungi (Additional file 3: Table S3 and Fig. 3a).
In M. anisopliae s.l., subtilisins were reported to degrade
host cuticles and allow acquiring nutrients, with potential
functional differences between subfamilies in secondary
substrate specificities and adsorption properties [37]. O.
polyrhachis-furcata,O. sinensis and C. militaris contain
about half the number of subtilisins (S08) compared with
B. bassiana,M. robertsii,andM. acridum.Elevenoutof
twenty-three subfamilies of subtilisins, were not identified
in any of the six entomopathogenic species while four are
common across all six species (Additional file 4: Table
S4). Among those that were commonly found in the
six insect fungi, subfamily S08.UPA (subfamily S8A
unassigned peptidases) is very abundant.
Regarding the subtilisins S53 (sedolisins), four out of five
subfamilies could be identified in entomopathogenic fungi.
O. polyrhachis-furcata and O. sinensis contain only one
protease in the subfamily S53.UPW (family S53 unassigned
peptidases), which is common across the six entomopatho-
genic fungi. Four other entomopathogenic species contain
a few more proteinases, including aorsin (S53.007) and gri-
folisin (S53.010) with one additional scytalidolisin (S53.011)
for C. militaris, which were not found in O. polyrhachis-
furcata and O. sinensis. The subfamily S53.003 (tripeptidyl-
peptidase I) was not identified in any six species.
The subtilisins (S08 and S53) of O. polyrhachis-furcata
derived from those of other entomopathogenic fungi do
OPSINE
OPF
METANI
METROB
BEUBAS
CORMIL
FUSGRA
BOTFT
BOTFW
SCLSCL
VERALB
NEUCRA
ASNIA
ASF
MAGORY
COPCIN
USTMAY
SCHIPO
SACCES
CANAlB
BATD8
PHI:2099
PHI:144
PHI:2403
PHI:409
PHI:1851
PHI:12
PHI:1209
PHI:1159
PHI:244
PHI:2328
PHI:2117
PHI:2190
PHI:355
PHI:698
PHI:251
PHI:231
PHI:315
PHI:1556
PHI:794
PHI:2464
PHI:2433
PHI:1714
PHI:1320
PHI:395
PHI:2434
PHI:1450
PHI:2430
PHI:1186
PHI:1364
PHI:653
PHI:2327
PHI:1008
PHI:578
PHI:1056
PHI:257
PHI:2323
PHI:1179
PHI:446
PHI:2432
PHI:1173
Fig. 2 A phylogram based on the number of genes in all PHI
families with presence/absence of some selected families. OPF =
Ophiocordyceps polyrhachis-furcata, OPSINE = Ophiocordyceps sinensis,
BEUBAS = Beauveria bassiana, CORMIL = Cordyceps militaris, METANI
=Metarhizium anisopliae, METROB = Metarhizium robertsii, FUSGRA =
Fusarium graminearum, VERALB = Verticillium alfalfae, SCLSCL =
Sclerotinia sclerotiorum, BOTFW = Botryotinia fuckeliana, ASNIA =
Aspergillus nidulans, ASF = Aspergillus fumigatus, SACCES =
Saccharomyces cerevisiae, CANAlB = Candida albicans, SCHIPO =
Schizosaccharomyces pombe, COPCIN = Coprinosis cinerea, USTMAY =
Ustilago maydis, BATD = Batrachochytrium dendrobatidis
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not form a monophyletic clade (Fig. 3a), indicating that they
have evolved independently from the subtilisins of other
entomopathagenic fungi. Five of them (OPF_4096,
OPF_2781, OPF_4408, OPF_1157, OPF_4650) could not be
assigned to any orthologous group with any other fungi.
These proteins were respectively annotated as S08.053
(MER006091:oryzin) (Aspergillus flavus), S08.UPA
(MER032872: subfamily S8A unassigned peptidases)
(M. grisea),S08.053 (MER087525:oryzin) (Aspergillus
clavatus), S08.056 (MER126013:cuticle degrading pep-
tidase of parasitic fungus) (Hirsutella minnesotensis), and
S08.056 (MER123475:cuticle degrading peptidase of para-
sitic fungus) (O. sinensis). However, it is to be noted that
orthologous groups of subtilisins inferred by Inparanoid/
QuickParanoids [38, 39] are not supported by the max-
imum likelihood-based phylogenetic inference (Fig. 3),
suggesting that the similarity-based method of Inpara-
noid/QuickParanoid should be taken with caution.
Both O. polyrhachis-furcata and O. sinensis contain only
one trypsin S01.UPB (subfamily S1B unassigned pepti-
dases), which is conserved across six entomopathogenic
fungi. Other insect fungi species have substantially higher
number of trypsins (22 in B. bassiana,34inM. robertsii,
15 in M. acridum and 12 in C. militaris). Subfamily
S01.UPA (subfamily S1A unassigned peptidases) was iden-
tified in all but the two Ophiocordyceps and the most abun-
dant family. Subfamily S01.412 (CHY1 peptidases or
chymotrypsins) was identified in B. bassiana (BBA_02727),
C. militaris (CCM_08282) and M. robertsii (MAA_07484)
but not in M. acridum. Additionally, only B. bassiana con-
tains additional two Nma111 peptidases (S01.434).
The numbers of aspartyl proteases (A01) identified in
O. polyrhachis-furcata and O. sinensis are about half of the
other four entomopathogenic fungi and in only four out of
thirty-five subfamilies. Subfamily A01.UPA (subfamily
A1A unassigned peptidases) is conserved across the six
bc
0
20
40
60
80
100
120
140
O.
polyrhachis-
furcata
O. sinensis C. mili taris M. acridum M. robertsii B. bassiana
Aspartyl proteases (A01)
Trypsin proteases (S01)
Subtilisin proteases (S53)
Subtilisin proteases (S08)
O. polyrhachis-furcata O. sinensis C. militaris M. acridum M. robertsii B. bassiana
Fig. 3 Expansions of proteases among six entomopathogenic fungi including O. polyrhachis-furcata.aThe distribution of proteases families among the
six entomopathogenic fungi included in this study. bA maximum likelihood-based unrooted phylogenetic tree illustrating the relation
between orthologous proteins ofsubtilisins(S08andS53).cA maximum likelihood-based unrooted phylogenetic tree illustrating the relation
between orthologous proteins of aspatyl proteases (A01). The sequences labels; colored following insect fungi species: red= O. polyrhachis-furcata
(OPF), brown = B. bassiana (BEUBAS), green = M. robertsii (METANI), dark blue = M. acridum (METACR), light blue = C. militaris (CORMIL), pink = O. sinensis
(OPSINE); are written in the form of cluster id._fungal species_sequences id. The cluster identities were obtained inferred according to Inparanoid/
QuickParanoid [38, 39]
Wichadakul et al. BMC Genomics (2015) 16:881 Page 6 of 14
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species and very abundant. Twenty-two out of thirty-five
subfamilies were not identified in any six species. Subfam-
ilies A01.018 (saccharopepsin) and A01.077 (CtsD peptid-
ase) were found in all but the two Ophiocordyceps species.
Several subfamilies A01.017 (endothiapepsin), A01.027
(trichodermapepsin), A01.057 (MernameAA034 peptid-
ase), A01.079 (PepAa peptidase), and A01.UPB (subfamily
A1B unassigned peptidases) were identified in only the
two Metarhizium species. Additionally, A01.082 (SA76
peptidase) was identified in all except O. sinensis (Add-
itional file 4: Table S4). Like subtilisins S08 and S53, the
aspatryl proteases of O. polyrhachis-furcata do not form a
monophyletic clade (Fig. 3b).
Like proteases, chitinases have been reported as in-
volved in host penetration which is a crucial first step of
pathogenesis and to influence the virulence [40–42].
The chitinases are classified into a family of Glycoside
Hydrolase (GH18). GH18 is the most abundant family of
Glycoside Hydrolase found in all fungi included in our
analysis (Additional file 5: Table S5). Among entomo-
pathogenic fungi, broad host-range species including B.
bassiana and M. robertsii also possess more genes en-
coding this GH family (195 and 179 respectively) than
M. acridum (160), C. militaris (157), O. sinensis (110)
and also O. polyrhachis-furcata (134). Additionnally, O.
polyrhachis-furcata and O. sinensis have less proteins of
several GH families than the other four entomopathogenic
fungi (GH16, GH3, GH4, GH76, GH92, GH43, GH78,
GH79: Additional file 5: Table S5). Some GH, and Polysac-
charide Lyase families were reported to be related to host-
specific adaptation in plant pathogens (i.e. GH6, GH7,
CBM1, GH28, GH78, GH88, GH95, PL1, PL3 [43]). All
six entomopathogenic fungi do not have the GH7 families,
less than half of the numbers of GH28, GH78, GH95
(Additional file 5: Table S5), and many less or no PL1 and
PL3 (Additional file 6: Table S6). Among these families;
GH28, GH78 showed the same tendency as that found for
GH18 in that broad host-range species possess more
genes than narrow host-range species including O.
polyrhachis-furcata. Regarding the families of carbohy-
drate esterases (CE), on one hand, O. polyrhachis-furcata
and O. sinensis have less number of CE for the families
CE1 and CE6, compared to the other four entomopatho-
genic fungi species. On the other hand, they contain simi-
lar number of proteins to the other insect fungi but
different to plant pathogens for the CE3, CE5 and CE12
families (Additional file 7: Table S7).
Bacterial-like toxins
Entomopathogenic fungi are expected to infect hosts
through cuticular penetration and thus to possess less
toxins compared with other pathogens such as bacteria
or viruses. However, the number of bacterial-like toxin
proteins and toxin-biosynthesis proteins found in
O. polyrhachis-furcata are relatively high (22), compared
to C. militaris (9), M. robertsii (34), M. acridum (10)
and O. sinensis (9); this number is actually the same as
B. bassiana [3] (Additional file 8: Table S8). M. robertsii
has the highest number of such proteins (34). Seventeen
out of twenty-two toxins found in the genome of O.
polyrhachis-furcata are heat-labile enterotoxins A chain.
Others include a zeta toxin, a ribotoxin, two cholera entor-
otoxins and a killer toxin. Among these toxins, a gene
putatively encoding a heat-labile A chain was identified in
front of a NRPS gene in a predicted NRPS gene cluster.
The prevalence of genes coding for bacterial-like toxins in
thegenomeofO. polyrhachis-furcata suggests the possi-
bility of oral toxicity as a mode of killing insect host.
Signal transduction
The encounter between the pathogens and the hosts will
inevitably engage the signal transduction from both parties.
We also found in the genome of O. polyrhachis-furcata
various genes related to cellular signal transduction includ-
ing G proteins, G protein-coupled receptors (GPCRs) and
histidine kinases (HK).
Among six entomopathogenic fungi, O. polyrhachis-
furcata contains the smallest number of Pth11-like G-
protein coupled receptors (GPCRs) which are cell-surface
integral membrane proteins required for pathogenicity
[44]. Also, as PHI genes (PHI-base accession: PHI:404 and
PHI:441), they mediate cell response to inductive cues [45].
PHI genes were shown to be related to host specificity of
M. acridum via differential expression on locust and
cockroach cuticles [2]. All other fourteen non-
entomopathogenic fungi clearly contain less number
of Pth11-like genes.
Two PHI:441 proteins in O. polyrhachis-furcata out of
seven, OPF_8495 (PHI:441|BTP1|CAE55153|TX:40559|-
Botrytis cinerea) and OPF_6721 (PHI:441|BTP1|CAE5
5153|TX:40559|Botrytis cinerea), have no orthologs
with any other nineteen compared species. Based on
BLASTX against NR database, the two proteins were
annotated as “hypothetical protein THITE_2110904”
and “putative integral membrane protein [Eutypalata
UCREL1]”. Meanwhile, six putative PHI:404 genes were
found in O. polyrhachis-furcata and could be clustered
into orthologous groups with the other fungi.
G protein alpha subunits have been comprehensively
studied, as a key component of signal transduction path-
ways and pathogenicity [46]. Its disruption in Stagonospora
nodorum madethefunguslesspathogenic,unableto
sporulate, and albino phenotype with secretion of brown
pigments into growth media [47]. In Metarhizium,G-alpha
proteins in M. robertsii (MAA_03488) and M. acridum
(MAC_04984) were observed as the most expressed
G-alpha genes during their infection of either cockroach or
locust cuticles [2]. These two genes are clustered together
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as orthologous proteins with all remaining eighteen
species including O. polyrhachis-furcata: OPF_3427. The
two-component Histidine Kinase (HK) signaling pathways
mediate environmental stress responses and regulation of
secondary metabolism [48, 49], response to bacterial me-
tabolites [50], hyphal development [51], virulence [52] and
sensitivity to dicarboximide and phenylpyrrole fungicides
[53, 54] in diverse fungal species. O. polyrhachis-furcata
contains the similar number of histidine kinases (9) com-
pared with M. robertsii (10) and M. acridum (9) reported
in [2].
Core genes involved in the biosynthesis of secondary
metabolites
Insect fungi are also well known for producing a variety of
secondary metabolites. Genes implicated in their biosyn-
thesis have received large attention, particularly polyke-
tides and non-ribosomal peptides synthetases (PKS and
NRPS) [6]. Based on SMURF [55], the O. polyrhachis-
furcata genome encodes 4 putative NRPS, other 4 NRPS-
like, 14 PKS, 1 PKS-like, 1 NRPS-PKS hybrid and without
dimethylallyl tryptophan synthase (DMAT) genes (Table 2).
The total number of 24 core genes in this species is much
less than that of M. robertsii (60) and M. acridum (42) [2]
and somewhat less than that of B. bassiana (36) [3] and
also C. militaris (28) [5]. Similar to M. robertsii, one of the
four putative NRPS-like genes in O. polyrhachis-furcata is
an antibiotic synthetase, reported for preserving the ca-
daver from microbial competitors [2]. Another NRPS-like
protein (OPF_1123) is mostly similar to another antibiotics
synthase (Linear gramicidin synthase subunit D of F. oxy-
sporum). The unique PKS/NRPS hybrid gene gets the top
hit to fusarin C cluster-polyketide synthase/NRPS, which
is a mycotoxin produced by several Fusarium species with
carcinogenic effects [56]. Beside these core genes, three
additional genes (fus2, fus8, and fus9), reported to be re-
sponsible for fusarin production [57], were also identified
within this hybrid cluster. O. polyrhachis-furcata possesses
an NRPS (OPF_4495), which is similar (39 % identity) to
peramine synthetase (PerA) of Epichloe festucae,theessen-
tial enzyme for the biosynthesis of peramine; a compelling
insect feeding deterrent to protect their grass host
from insect herbivory [58]. Based on antiSMASH [59],
O. polyrhachis-furcata also possesses the PKS-like and
NRPS-like which are respectively similar to Ochratoxin A
polyketide synthase (35 % identity) and a non-ribosomal
peptide synthetase (36 % identity) of Penicillium nordicum
characterized in [60]. Even located on the same scaffold,
they are not on the same cluster.
Repeat elements and transposases
On one hand, the genome of O. polyrhachis-furcata
comprises notably more Class II transposable elements
(TEs) - the DNA transposons including DNA/hAT (23),
DNA/Mariner (7), DNA/MuDR (11), and DNA/Helitron
(7) - compared with the genomes of C. militaris and M.
acridum which contain less than five of these elements
in their genomes. On the other hand, O. polyrhachis-
furcata possess less of these elements compared to the
genomes of B. bassiana and M. robertsii (Fig. 4 and see
Additional file 9: Table S9) except for DNA/Helitron of
which B. bassiana is devoid. Particularly, M. robertsii
contains several more DNA/Helitron (26) compared
with O. polyrhachis-furcata (9) and M. acridum (5)
while C. militaris do not have this specific element. Re-
garding the Class I elements or the retroelements, the
Table 2 Number of core genes involved in the biosynthesis of secondary metabolites
Fungi DMAT HYBRID NRPS NRPS-like PKS PKS-like Total
O. polyrhachis-furcata 0 1 4 4 14 1 24
C. militaris 13 58 92 28
M. acridum 3 1 13 8 13 4 42
M. robertsii 5 5 14 9 24 3 60
B. bassiana 0 3 13 7 12 1 36
B. cinerea 1 0 6 8 16 6 37
S. sclerotiorum 1 0 5 5 16 2 29
F. graminearum 0 1 10 11 14 1 37
E. festucae 4 1 18 9 11 3 46
N. crassa 10 33 62 15
M. oryzae 3 3 5 6 12 3 32
A. nidulans 6 1 11 12 24 4 58
A. fumigatus 7 1 13 5 13 1 40
This table is extended and rearranged from Table S13 in [2]byaddingO. polyrhachis-furcata’s identified core genes following SMURF [55]. Core genes of C. militaris and B.
bassiana were exce rpted from Ta ble 3 in [5] and Table 2 in [3], respectively. DMAT: Dimethylallyl tryptophan synthase, NRPS: non-ribosomal peptide synthetase, HYBRID,
hybrid PKS–NRPS enzyme
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LTR/Copia, LTR/Gypsy, as well as the Non-LTR/LINE are
much more abundant in O. polyrhachis-furcata compared
to those species. Nevertheless, except for DNA/hAT and
DNA/helitron, O. sinensis contain many more transpos-
able elements than the other insect fungi species studied
here (Additional file 9: Table S9). Transposases were re-
ported to be expressed during infection in Metarhizium
[2]. Therefore, this type of genomic mobile element may
be related to pathogenicity.
Discussion
We present here a genome draft of an outstanding in-
sect fungus, O. unilateralis s.l. from the host Polyrha-
chis furcata. In our study, we compare this genome to
other available fungal genomes of various origins in
order to gain insights into the biology of this species.
The particularity of this fungus is its extreme host-
specificity as shown by the fact that different species
are associated to different ant species [10, 11]. Here, we
have sequenced O. polyrhachis-furcata which is specific
to an ant species while, for the others, the degree of
host range is variable, ranging from families of the same
insect orders (e.g. O. sinensis and O. militaris)tosev-
eral insect orders (e.g. B. bassiana,M. robertsii).
We found that the genome of O. polyrhachis-furcata
contains various genes which were shown to be impli-
cated during the pathogenesis of fungal pathogens. Many
of them are in common with other entomopathogenic
fungi and fungal plant pathogens.
In this genome, we observed contractions of several
kinds of genes, compared to broad host range entomo-
pathogenic fungi. This signature of genes contraction is
found for genes involved throughout the pathogenesis,
from genes required for the adhesion of infective propa-
gules to the synthesis of metabolites potentially neces-
sary for coping with insect immune systems. This may
have critical implications in the life of this fungus. For
example, hydrophobins Classes 1 and 2 are amphiphilic
proteins, mostly involved in the mediation of the contact
between the fungal cells and hydrophobic/hydrophilic
surfaces. Although the roles of both classes and different
paralogs are not yet totally elucidated and partially over-
lapping [31], the disruption of a number of hydropho-
bins resulted in altered hydrophobicity of the mycelia as
well as the morphology and production of conidia [33–
35]. The absence of hydrophobin Class 1 in O.
polyrhachis-furcata could be related to fastidious growth
and sporulation of this species in standard complex
media while B. bassiana,M. spp.,C. militaris and O.
sinensis are all fast growers and can produce spores
abundantly in the laboratory.
Comparison with extensive available fungal genomes
allowed us to grasp common aspects and differences be-
tween plant and insect pathogens. A notable difference
is the absence of MAD2 adhesin in plant pathogens,
which is not the case for the entomopathogenic fungi
except O. sinensis. This suggests that the entomopatho-
genic fungi may have dual ways of life, being parasitic to
insects and in the same time having the capacity to be
endophytes in plants. This has already been shown in B.
bassiana and M. anisopliae [61, 62]. The absence of
MAD2 in plant pathogens suggests a totally different
mechanism of adhesion. Furthermore, notable differences
in numbers of genes are found for various Glycoside
Hydrolase (GH), Carbohydrate Esterase (CE) families
as well as Polysaccharide Lyase (PL) families. Over-
all, the comparative analysis shows a cleavage be-
tween insect and plant pathogens in the penetration
of host’s cell. This may be linked to the fact that
fungal pathogens of plants need to overcome the cell
wall which has different biochemical composition to
insect cuticle.
Subtilisin proteases have been shown to play crucial role
in regulating the specificity to hosts of entomopathogenic
0
20
40
60
80
100
120
140
hAT Mariner MuDR Helitron Copia Gypsy NonLTR/LINE
B. bassiana C. militaris M. robertsii M. acridum O. polyrhachis-furcat
a
Fig. 4 The numbers of transposases between the genomes of different entomopathogenic fungi. The data of O. sinensis [4] were not included in this
graphic as this species contain much higher numbers of most transposable elements classes (Additional file 9: Table S9) and skewed the representation
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fungi via the reduction of isoforms [37] or the differential
expressions of isoforms according to substrates [63]. Hosts
play important role in the evolution of subtilisin-like pro-
teases in entomopathogenic fungi by imposing constraints
into the realized niches in vivo.InMetarhizium, generalist
strains were shown to have more paralogous isoforms
than a specialist strain [37]. According to our comparative
analysis, there is also a tendency in the reduction of subtil-
isins proteases genes for narrow-host range species of in-
sect fungi with O. polyrhachis-furcata having in effect the
narrowest host range and one of the lowest numbers of
genes coding for subtilisins proteases compared with the
other entomopathogenic fungi. This is in accordance with
the pattern of the reduction in genes number, found in
GH and CE as well as in other genes coding for cuticle-
degrading proteolytic enzymes like trypsin and aspatyl
proteases, for narrow-host range species. This suggests a
direct association between host range and diversity of
cuticle-degrading enzymes. Altogether, this consistently
supports the early host recognition events as key to host
specificity and the tendency toward reduced genes num-
bers associated with narrow host range.
Referred to the PHI genes classification, we observed
also that O. polyrhachis-furcata and O. sinensis had sub-
stantially less genes for some families when compared to
the other entomopathogenic fungi. One of these families
is clearly related to the membrane transport of sucrose
(PHI:2240). The difference in number of genes for this
family may result from the affinity to different nature of
carbon sources in different insect hosts. Another family
of PHI genes (PHI:511), which has different numbers of
genes between O. polyrhachis-furcata/O. sinensis and
the broad host range species, is involved in drug sensi-
tivity. Systemic immune response in different hosts may
influence on the evolution of this family of genes in en-
tomopathogenic fungi. Other families of PHI genes
showing substantial reduction of gene numbers in O.
polyrhachis-furcata are PHI:404 and PHI:441 which are
also Pth11-like G-protein coupled receptors (GPCRs)
previously shown to be related to host specificity. O.
polyrhachis-furcata has the lowest number of Pth11-like
genes compared to other five entomopathogenic fungi.
The genes contraction in these various families suggest
that specific species like O. polyrhachis-furcata may have
lost a plethora of proteins of these particular families
which potentially cope with diverse systemic immune re-
sponses from unspecific host.
An unexpectedly high number of genes related to biosyn-
thesis of bacterial-like toxins are found in the genome of O.
polyrhachis-furcata, compared to other entomopathogenic
fungi species considered as having narrow host range (M.
acridum,C. militaris). Previously, comparative genomics
between generalist and specialist strains in M. anisopliae
s.l. (subsequently M. robertsii and M. acridum)[64]
indicated that specialists had reductive evolution of genes
involved in toxin biosynthesis, suggesting that they had lost
the capacity to kill host rapidly and to exploit host sapro-
trophically. Instead, they got specialized into strategies opti-
mizing the struggle against host immune system and
exploitation of living hosts. Furthermore, our comparative
analysis also supports this hypothesis regarding M. robertsii
and M. acridum which differ in their host ranges and diver-
sity of putative genes producing toxins. Therefore, we ex-
pect O. polyrhachis -furcata which is an extreme specialist
to have evolved into the latter strategy while the data sup-
port for the former. This suggests that O. unilateralis may
have a totally different mechanism for killing host from that
of M. anisopliae. Toxins found in O. polyrhachis-furcata’s
genome are for many enterotoxins, suggesting that oral
toxicity may constitute a mode of killing for our fungus.
However, how O. polyrhachis-furcata, as a species of O.
unilateralis which is well documented for its extended phe-
notypes in manipulating the ant host, strives to comprom-
ise between rapid killing by toxin and slow killing enabling
the host manipulation remains to be solved.
Another distinctive feature of O. polyrhachis-furcata’s
genome is the composition of transposable elements.
The LTR/Copia and LTR/Gypsy are much more abun-
dant in O. polyrhachis-furcata than in the other insect
fungi except O. sinensis which has the highest numbers
of transposable elements of almost all classes. It is well
known that transposable elements are important source
of genetic variability of different species and also con-
tribute to intra-specific diversification in various fungi
[65]. Also, transposable elements were shown to have
important roles in pathogenicity and virulence for many
microorganisms. Particularly, retrotransposons were
shown be involved in the pathogenicity of C. albicans
[66] and associated with the rapid evolution of effectors
and the adaptation to new host in other pathogenic fila-
mentous fungi [67] and Oomycetes [68]. For insect
fungi, it was shown that more than 65 % of the transpo-
sase genes were transcribed in Metarhizium hyphae dur-
ing the infection process [2]. Therefore, the enrichment
of retrotransposons in O. polyrhachis-furcata and O.
sinensis could be related to their high host specificity
and a unique pathogenicity compared to the other insect
fungi. The evolution of host specificity and the diversifi-
cation of insect fungi may thus be driven by the expan-
sion/reduction of these elements.
Conclusions
The sequencing and annotation of fungal genomes have
allowed mycologists to gain insight into the fundamental
aspects of fungal and eukaryotic biology. Comparative
analyses between O. polyrhachis-furcata and some other
fungi give unprecedented insights into the biology of this
species, particularly regarding the emergence of its
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extreme host specificity. Gene contractions have been pos-
tulated as being one of major causes in the evolution of
host-specificity in various organisms. This is due to the
fact that losses of genes are accompanied by losses of cap-
acity of the organisms to exploit range of hosts. The con-
traction of various gene families in O. polyrhachis-furcata
is in line with this hypothesis. However, this cannot alone
explain the evolution of host specificity. Specific genes and
even genomic regions or chromosomes are also the origin
of host specificity in many fungal pathogens. Some genes
found in O. polyrhachis-furcata are unique among ento-
mopathogenic fungi. The contribution of these genes to
the unique biology and host specificity of O. unilateralis
s.l. remains to be studied. Particularly, the expansions of
genes related to the production of bacterial-like toxins and
of retrotransposons are of major interests. While loss of
toxins and capacity to kill hosts rapidly was proposed as
on mechanism underlying host-specificity [64], the pattern
of expansion observed in O. polyrhachis-furcata suggest a
completely different mechanism. Also, the expansion of
retrotransposons in this fungus is unique among insect
fungi. These findings will pave a way to a better under-
standing of this unique organism and provide promising
field of research.
Methods
Fungal strains
O. polyrhachis-furcata (BCC54312) was collected from
Khao Yai National Park in Nakhon Ratchasima province
of Thailand. The strain was isolated and grown on Grace
Insect Cell Medium and PDA according to [69]. After
the growth on PDA for two months, the mycelia were
harvested separated into several eppendorfs, not to ex-
ceed approximately 100 mg per tube. The mycelia were
extracted for DNA by DNeasy Plant Mini Kit (QIAGEN)
by following the manual for 454 sequencing and also
using a modified CTAB extraction for samples aimed for
mate-pair sequencing.
Genome/transcriptome sequencing and assembly
The genomic DNA sample of Ophicordyceps polyrhachis-
furcata was extracted and used to construct shotgun gen-
omic DNA library according to the GS FLX protocol
(Roche). The shotgun reads were de novo assembled by
Newbler v.2.8. In order to construct scaffolds, the gen-
omic DNA sample was used to construct mate-pair librar-
ies with varied insertion sizes of 3, 6 and 8 kb sequencing
which were sequenced on Hiseq2000 (Illumina). The
mate-pair reads together with the pre-assembled contigs
were then used for scaffolding using SSPACE 2.0 [70].
To obtain the transcriptome, O. polyrhachis-furcata
(BCC54312) was grown on four different media formula-
tions: PDA, PDA + Bacto Soytone 1 %, PDA + Malt Ex-
tract 0.5 % and PDA + Phyto Peptone 2 %. The mycelia
from different media were harvested together and frozen
with liquid nitrogen until the RNA extraction. Frozen
mycelia were grinded and transferred to 2.0 ml tubes.
1 ml of RNA extraction reagent (Invitrogen) was added
to each tube, vortexed and added 0.1 ml 5 M NaCl and
0.3 ml Chloroform. The solutions were then centrifuged
at 14,000 rpm for 5 min at 4 °C. The supernatants were
then transferred to new tubes and added equal volumes
of Chloroform (~1 ml), vortexed and centrifuged at
14,000 rpm for 10 min at 4 °C. The chloroform extrac-
tion was repeated until there was not interphase any-
more. The RNA was precipitated with cold ethanol and
3 M LiCl, incubated at −20 °C overnight. The tubes were
then centrifuged at 12,000 rpm for 25 min at 4 °C. The
RNA pellets were washed with 70 % ethanol twice and
re-suspended in 50 ul DEPC-water.
mRNA (200 ng) was isolated from the total RNA sam-
ple using mRNA isolation kit (Stratagene) and subjected
to cDNA library construction and sequencing on the GS
FLX platform (Roche). We obtained 568,0911 reads with
the average read length of 538 bp. The cDNA reads were
cleaned (trim poly-A tail, remove short/repetitive/low-
quality reads) by SeqClean [http://sourceforge.net/pro-
jects/seqclean/] and 535,728 reads were de novo assem-
bled into 12,293 contigs by Newbler cDNA de novo
assembler (Roche). The transcriptome was used to im-
prove the annotation.
Gene prediction and annotation
MAKER (v.2.28) [71] was used as the main annotation
pipeline for gene prediction and annotation. As part of
MAKER, GlimmerHMM (v.3.0.1) [72], AUGUSTUS
(v.2.6.1) [73, 74], SNAP (released 29/11/2013) [75], and
GeneMark-ES fungal version [76] were used as ab initio
gene predictors. The ab initio SNAP was trained for a
couple times within MAKER based on sequence similar-
ity search via BLAST [77] and sequence alignment via
Exonerate (v.2.2.0) [78] with default parameter settings,
using 386,567 EST and 248,253 protein sequences of the
order Hypocreales compiled from NCBI in October
2013, together with 10,848 assembled RNA-Seq tran-
scripts, against the 598 scaffolds which were also masked
by RepeatMasker (v.4.0.3) [79] together with Tandem
Repeat Finder (TRF) (v.4.04) [80] and RMBlast (NCBI
blast package of RepeatMasker), based on Repbase data-
base (repeatmaskerlibraries-20130422.tar.gz; [81]). The
transposable elements were extracted from the various
types of masked repeats. The predicted transcripts were
searched against the non-redundant protein database
(NR) downloaded from NCBI on Feb 10, 2014. The
BLAST results were then imported into BLAST2GO
[82, 83] for Gene Ontology (GO), Enzyme, and KEGG
pathway annotations. The tRNAs were predicted by the
tRNAscan-SE (v.1.3.1) [84] and Aragorn (v.1.2.34) [85]
Wichadakul et al. BMC Genomics (2015) 16:881 Page 11 of 14
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and the protein secretion was predicted by SignalP 4.0
[12]. The completeness of genome assembly and gene
annotation was assessed using BUSCO (v.1.1b1) [86],
with the 598 scaffolds and predicted protein sequences
of O. polyrhachis-furcata as respective inputs and the
BUSCO’s fungal dataset of 1438 benchmarking universal
single-copy orthologs as profile.
Orthology and phylogenomic analysis
The standalone InParanoid (v.4.1) [38] and QuickParanoid
[39] with default parameter settings were respectively used
to identify and cluster the orthologous proteins of O.
polyrhachis-furcata and the nineteen other taxa (the pro-
teomes of these taxa were downloaded from UniProt:
Additional file 10: Table S10). The concatenated 1,053
protein sequences conserved among all taxa were used as
input of RAxML (v.8) [87] for building the phylogenomic
tree using the Dayhoff model.
Protein family classifications
The potential pathogenic and virulence genes were iden-
tified by BLASTP against the pathogen-host interaction
database (PHI-base) v.3.5 [13]. The families of proteases
were identified by BLASTP against the peptidase data-
base (MEROPS) release 9.10 [88, 89] with the E-value
cutoff < = 1E-20. The carbohydrate active enzymes;
glycoside hydrolases (GHs), polysaccharide lyases (PLs),
carbohydrate esterases (CEs), were classified by the
CAZy database [90], of which protein sequences were
manually compiled via a CAZy tool [91] kindly provided
by Alexander Holm Viborg. The G-protein coupled recep-
tors and transporters were classified based on BLASTP
against the GPCRDB [92] (version 2013.09.26) and Trans-
porter Classification Database (TCDB) (Last modified: July
15, 2011) [93], respectively. BLASTP against all specific
protein databases except MEROPS used E-value cutoff < =
1E-5. Gene clusters and core genes for biosynthesis of sec-
ondary metabolites were identified by SMURF [55] and
antiSMASH [94]. For phylogenetic analysis, sequences of
all species hit with specific protein family (i.e., MEROPS:
S08 and S53 for subtilisins) were extracted and multiple
aligned using MAFFT v.7 [95]. RAxML version 8 [87] was
then used to analyze the phylogenetic trees based on max-
imum likelihood and visualized by Dendroscope 3 [96].
Additional files
Additional file 1: Table S1. The number of genes for PHI genes family
identified in O. polyrhachis-furcata and nineteen other fungi. (XLSX 252 kb)
Additional file 2: Table S2. The blast results for the protein-coding
genes of O. polyrhachis-furcata against PHI database. (XLS 385 kb)
Additional file 3: Table S3. The number of subtilisins, trypsins, and
aspartyl proteases identified in O. polyrhachis-furcata compared with
nineteen other fungi. (XLSX 14 kb)
Additional file 4: Table S4. The number of genes following the MEROPS
databases (peptidases) identified in O. polyrhachis-furcata,comparedwith
the nineteen other fungi included in this study. (XLSX 50 kb)
Additional file 5: Table S5. The number of genes in different Glycoside
Hydrolase (GH) families, following the Carbohydrate-Active Enzymes Databases
(CAZY), identified in O. polyrhachis-furcata and other nineteen fungi included
in this study. (XLSX 21 kb)
Additional file 6: Table S6. The number of genes in different
Polysaccharide Lyase (PL) families, following the Carbohydrate-Active
Enzymes Databases (CAZY), identified in O. polyrhachis-furcata and
other nineteen fungi included in this study. (XLSX 10 kb)
Additional file 7: Table S7. The number of genes in different
Carbohydrate Esterase (CE) families, following the Carbohydrate-
Active Enzymes Databases (CAZY), identified in O. polyrhachis-furcata
and other nineteen fungi included in this study. (XLSX 10 kb)
Additional file 8: Table S8. The number of bacterial-like toxins identi-
fied in O. polyrhachis-furcata compared with other entomopathogenic
fungi. (XLSX 15 kb)
Additional file 9: Table S9. The number of transposable elements (TEs)
identified in OPF compared with other entomopathogenic fungi. The data
of B. bassiana,C. militaris,M. robertsii,andM. acridum were obtained from
Supplemental Table S2 of [3]. The data of M. anisopliae was obtained from
[2]. The data of O. sinensis was obtained from [4]. (PDF 10 kb)
Additional file 10: Table S10. List of nineteen fungal species selected
from UniProt for orthology and comparative analysis with O. polyrhachis-
furcata BCC54312. (PDF 29 kb)
Competing interests
The authors declare that they have no competing interests.
Authors’contribution
DW carried out the annotation, analyses and discussion of the data, as well as
drafting of the manuscript. NK carried out the collection of strains, molecular
work and drafting of the manuscript and interpretation of the data. SI carried out
the partial analyses of data and help in the annotation as well as discussion of
the results and drafting the manuscript. ST carried out the genome sequencing
and discussion of the results and drafting of the manuscript. DC carried out the
transcriptomics work to improve the annotation. JJL conceived and designed the
study, carried out the collection of strains, participated in its design and helped
draft the manuscript. LE participated in the design of the study, coordination and
discussion of the results. All authors have participated sufficiently in the
completion of the work, have read and approved the final manuscript.
Acknowledgements
This work was supported by the Platform Technology Management Section
(grant number P-12-01036), National Center for Genetic Engineering and
Biotechnology (BIOTEC), National Science and Technology Development
Agency (NSTDA), Thailand. Mrs. Rachada Promhan is thanked for growing
the fungus and maintaining the culture, Mrs. Suchada Mongkolsamrit
and Ms. Kanoksri Tasanathai are thanked for their help in collecting the
fungus. We also would like to thank the anonymous reviewers for their
suggestions and comments to improve the manuscript.
Author details
1
National Center for Genetic Engineering and Biotechnology, National
Science and Technology Development Agency, 113 Thailand Science Park,
Phahonyothin Rd., Khlong Neung, Khlong Luang 12120 Pathum Thani,
Thailand.
2
Department of Computer Engineering, Faculty of Engineering,
Chulalongkorn University, Floor 17th, Building 4, Payathai Rd., Wangmai,
Pathumwan 10330 Bangkok, Thailand.
Received: 13 July 2015 Accepted: 16 October 2015
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