RESEARCH Open Access
Comparative genome sequence analysis
underscores mycoparasitism as the ancestral life
style of Trichoderma
Christian P Kubicek1*, Alfredo Herrera-Estrella2, Verena Seidl-Seiboth1, Diego A Martinez3, Irina S Druzhinina1,
Michael Thon4, Susanne Zeilinger1, Sergio Casas-Flores5, Benjamin A Horwitz6, Prasun K Mukherjee7,
Mala Mukherjee6, László Kredics8, Luis D Alcaraz2, Andrea Aerts9, Zsuzsanna Antal8, Lea Atanasova1,
Mayte G Cervantes-Badillo5, Jean Challacombe9, Olga Chertkov9, Kevin McCluskey10, Fanny Coulpier11,
Nandan Deshpande12, Hans von Döhren13, Daniel J Ebbole14, Edgardo U Esquivel-Naranjo2, Erzsébet Fekete15,
Michel Flipphi16, Fabian Glaser6, Elida Y Gómez-Rodríguez5, Sabine Gruber1, Cliff Han9, Bernard Henrissat17,
Rosa Hermosa4, Miguel Hernández-Oñate2, Levente Karaffa15, Idit Kosti6, Stéphane Le Crom11, Erika Lindquist9,
Susan Lucas9, Mette Lübeck18, Peter S Lübeck18, Antoine Margeot19, Benjamin Metz1, Monica Misra9,
Helena Nevalainen12, Markus Omann1, Nicolle Packer12, Giancarlo Perrone20, Edith E Uresti-Rivera5, Asaf Salamov9,
Monika Schmoll1, Bernhard Seiboth1, Harris Shapiro9, Serenella Sukno4, Juan Antonio Tamayo-Ramos21,
Doris Tisch1, Aric Wiest10, Heather H Wilkinson14, Michael Zhang9, Pedro M Coutinho17, Charles M Kenerley14,
Enrique Monte4, Scott E Baker9,22 and Igor V Grigoriev9
Abstract
Background: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance
when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection.
Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.
Results: Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride
(teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens),
and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display
a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to
repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T.
reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis
shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose
in a common Trichoderma ancestor but were subsequently lost in T. reesei.
Conclusions: The data offer a better understanding of mycoparasitism, and thus enforce the development of
improved biocontrol strains for efficient and environmentally friendly protection of plants.
* Correspondence: ckubicek@mail.zserv.tuwien.ac.at
1Area Gene Technology and Applied Biochemistry, Institute of Chemical
Engineering Vienna University of Technology, Getreidemarkt 9, 1060 Vienna,
Austria
Full list of author information is available at the end of the article
Kubicek et al. Genome Biology 2011, 12:R40
http://genomebiology.com/2011/12/4/R40
© 2011 Kubicek 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.

Background
Mycoparasitism is the phenomenon whereby one fungus
is parasitic on another fungus, a lifestyle that can be
dated to at least 400 million years ago by fossil evidence
[1]. This has special relevance when the prey is a plant
pathogen, providing a strategy for biological control of
pests for plant protection (’biocontrol’). The movement
toward environmentally friendly agricultural practices
over the past two decades has thus accelerated research
in the use of biocontrol fungi [2]. Probably the most stu-
died biocontrol agents are species of the genus Hypocrea/
Trichoderma, Trichoderma atroviride (Ta) and Tricho-
derma virens (Tv) - teleomorphs Hypocrea atroviridis
and Hypocrea virens, respectively - being among the best
mycoparasitic biocontrol agents used in agriculture [3].
The beneficial effects of Trichoderma spp. on plants
comprise traits such as the ability to antagonize soil-
borne pathogens by a combination of enzymatic lysis,
secretion of antibiotics, and competition for space and
substrates [4,5]. In addition, it is now known that some
Trichoderma biocontrol strains also interact intimately
with plant roots, colonizing the outer epidermis layers,
and acting as opportunistic, avirulent plant symbionts [6].
Science-based improvement of biocontrol agents for
agricultural applications requires an understanding of
the biological principles of their actions. So far, some of
the molecular aspects - such as the regulation and role
of cell wall hydrolytic enzymes and antagonistic second-
ary metabolites - have been studied in Trichoderma
[3-5]. More comprehensive analyses (for example, by
the use of subtractive hybridization techniques, proteo-
mics or EST approaches) have also been performed
with different Trichoderma species, but the interpreta-
tion of the data obtained is complicated by the lack of
genome sequence information for the species used
(reviewed in [7]).
Recently, the genome of another Trichoderma, Tricho-
derma reesei (Tr, teleomorph H. jecorina), which has a
saprotrophic lifestyle and is an industrial producer of
plant biomass hydrolyzing enzymes, has been sequenced
and analyzed [8]. Here we report the genome sequen-
cing and comparative analysis of two widely used bio-
control species of Trichoderma, that is, Ta and Tv.
These two were chosen because they are distantly
related to Tr [9] and represent well defined phylogenetic
species [10,11], in contrast to Trichoderma harzianum
sensu lato, which is also commonly used in biocontrol
but constitutes a complex of several cryptic species [12].
Results
Properties of the T. atroviride and T. virens genomes
The genomes of Ta IMI 206040 and Tv Gv29-8 were
sequenced using a whole genome shotgun approach to
approximately eight-fold coverage and further improved
using finishing reactions and gap closing. Their genome
sizes were 36.1 (Ta) and 38.8 Mbp (Tv), and thus larger
than the 34 Mbp determined for the genome of Tr [8].
Gene modeling, using a combination of homology and
ab initio methods, yielded 11,865 gene models for Ta
and 12,428 gene models for Tv, respectively (Table 1),
both greater than the estimate for Tr (9,143). As shown
in Figure 1, the vast majority of the genes (7,915) occur
in all three Trichoderma species. Yet Tv and Ta contain
about 2,756 and 2,510 genes, respectively, that have no
true orthologue in any of the other species, whereas Tr
has only 577 unique genes. Tv and Ta share 1,273
orthologues that are not present in Tr, which could thus
be part of the factors that make Ta and Tv mycopara-
sites (for analysis, see below).
With respect to other ascomycetes, Tr, Ta and Tv
share 6,306/7,091, 6,515/7,549, and 6,564/7,733 ortholo-
gues with N. crassa and Gibberella zeae, respectively.
Table 1 Genome assembly and annotation statistics
T. atroviride T. virens T. reesei
Genome size, Mbp 36.1 38.8 34.1
Coverage 8.26× 8.05× 9.00×
Assembly gaps, Mbp 0.1 (0.16%) 0.2(0.4%) 0.05 (0.1%)
Number of scaffolds 50 135 89
Number of predicted genes 11865 12518 9143
Gene length, bp 1747.06 1710.05 1793,25
Protein length, amino acids 471.54 478.69 492,27
Exons per gene 2,93 2,98 3,06
Exon length, bp 528.17 506.13 507,81
Intron length, bp 104.20 104.95 119,64
Supported by homology, NR 10,219 (92%) 10,915 (94%) 8409 (92%)
Supported by homology, Swissprot 8,367(75%) 8,773 (75%) 6763 (74%)
Has PFAM domain 5,883 (53%) 6,267 (54%) 5096 (56%)
NR, non-redundant database; PFAM, protein families.
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Thus, approximately a third of the genes in the three
Trichoderma species are not shared in even the rela-
tively close relative G. zeae and are thus unique to
Trichoderma.
Genome synteny
A comparison of the genomic organization of genes in
Ta, Tv and Tr showed that most genes are in synteny:
only 367 (4%) genes of Tr, but 2,515 (22%) of genes of
Tv and 2,690 (21%) genes of Ta are located in non-
syntenic regions (identified as a break in synteny by a
series of three or more genes (Table 2); a global visual
survey can be obtained at the genome websites of the
three Trichoderma species (see Materials and methods)
by clicking ‘Synteny’ and ‘Dot Plot’). As observed for
other fungal genomes [13-15], extensive rearrangements
have occurred since the separation of these three fungi
but with the prevalence of small inversions [16]. The
numbers of the synteny blocks increased with their
decreased size, compatible with the random breakage
model [14] as in aspergilli [15,17]. Sequence identity
between syntenic orthologs was 70% (Tr versus Ta),
78% (Tr versus Tv), and 74% (Tv versus Ta), values that
are similar to those calculated for aspergilli (for exam-
ple, Aspergillus fumigatus versus Aspergillus niger (69%)
and versus Aspergillus nidulans (68%) and comparable
to those between fish and man [17,18].
Transposons
A scan of the genome sequences with the de novo
repeat finding program ‘Piler’ [19] - which can detect
repetitive elements that are least 400 bp in length, have
more than 92% identity and are present in at least three
copies - was unsuccessful at detecting repetitive ele-
ments. The lack of repetitive elements detected in this
analysis is unusual in filamentous fungi and suggests
that, like the Tr genome [8], but unlike most other fila-
mentous fungi, the Ta and Tv genomes lack a signifi-
cant repetitive DNA component.
Because of the paucity of transposable elements (TEs)
in the Trichoderma genomes, we wondered whether
simple sequence repeats and minisatellite sequences
may also be rare. To this end, we surveyed the genomes
of the Trichoderma species using the program Tandem
Repeat Finder [20]. We also included the genomes of
three additional members of the Sordariomycetes and
one of the Eurotiomycetes as reference (Table S1 in
Additional file 1). Satellite DNA content varied from as
little as 2,371 loci (0.53% of the genome) in A. nidulans
to 9,893 (1.46% of the genome) in Neurospora crassa.
Satellite DNA content of the Trichoderma genomes ran-
ged from 5,249 (0.94%) in Ta to 7,743 (1.54%) in Tr.
Since these values are within the range that we found in
the reference species, we conclude that there is no unu-
sual variation in the satellite DNA content of the Tri-
choderma genomes.
We also scanned the genomes with RepeatMasker and
RepeatProteinMask [21] to identify sequences with simi-
larity to known TEs from other organisms. Thereby,
sequences with significant similarity to known TEs from
other eukaryotes were identified (Table 3). In most
cases, the TE families that we detected were fragmented
and highly divergent from one another, suggesting that
they did not arise from recent transposition events.
Table 2 Occurrence of orthologues, paralogues and
singletons in the genomes of the three Trichoderma spp
Genome Synteny Total
genes
Orthologsa Non-
orthologs
P-
valueb
T.
atroviride
Syntenic 9,350 7,326 2,024 2.2e-16
Non-
syntenic
2,515 1,265 1,250
T. virens Syntenic 9,828 7,326 2,502 2.2e-16
Non-
syntenic
2,690 1,532 1,158
T. reesei Syntenic 8,776 7,326 1,450 2.2e-16
Non-
syntenic
367 153 214
aOrthologs that are in all three genomes. bNull hypothesis that the proportion
of non-orthologs that are syntenic is less than the proportion of non-
orthologs that are non-syntenic. P-value: null hypothesis that the proportion
of paralogs that are syntenic is less than the proportion of paralogs that are
non-syntenic.
T. virens
T. reesei T. atroviride
7 915
484
167
1 273
2 510
2 756
577
Figure 1 Distribution of orthologues of T. atroviride, T. virens
and T. reesei. The Venn diagram shows the distribution found for
the three species of Trichoderma.
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Based on these results, we conclude that no extant,
functional TEs exist in the Trichoderma genomes. The
presence of ancient, degenerate TE copies suggests that
Trichoderma species are occasionally subject to infec-
tion, or invasion by TEs, but that the TEs are rapidly
rendered unable to replicate and rapidly accumulate
mutations.
Evidence for the operation of repeat-induced point
mutation in Trichoderma
The paucity of transposons in Trichoderma could be
due to repeat-induced point mutation (RIP), a gene
silencing mechanism. In N. crassa and many other fila-
mentous fungi, RIP preferentially acts on CA dinucleo-
tides, changing them to TA [22]. Thus, in sequences
that have been subject to RIP, one should expect to find
a decrease in the proportion of CA dinucleotides and its
complement dinucleotide TG as well as a corresponding
increase in the proportion of TA dinucleotides. The RIP
indices TA/AT and (CA + TG)/(AC + GT) developed
by Margolin et al. [22] can be used to detect sequences
that have been subject to RIP. Sequences that have been
subjected to RIP are expected to have a high TA/AT
ratio and low (CA + TG)/(AC + GT) ratio, with values
>0.89 and <1.03, respectively, being indicative of RIP
[22,23].
To identify evidence for RIP in the TE sequences, we
computed RIP indices for four of the most prevalent
TE families in each of the three species (Table 4).
Since many of the sequences are very short, we com-
puted the sum of the dinucleotide values within each
TE family within each species, and used the sums to
compute the RIP ratios. In only one of the 12 families
did we find that both RIP indices were within the
ranges that are typically used as criteria for RIP. Most
of the TE sequences that we identified in the Tricho-
derma genomes are highly degenerate and have likely
continued to accumulate mutations after the RIP pro-
cess has acted on them. We suspect that these muta-
tions have masked the underlying bias in dinucleotide
frequencies, making the RIP indices ineffective at iden-
tifying the presence of RIP. To overcome this, we also
prepared manually curated multiple sequence align-
ments of the TE families, selecting only sequences that
had the highest sequence similarity, and thus should
represent the most recent transposon insertion events
in the genomes. We were able to prepare curated
alignments for all four of the test TE families of Tr
and Tv only for the long terminal repeat element
Gypsy and the long interpersed nuclear element R1 in
Ta (Table S2 in Additional file 1). Among DNA
sequences that make up these ten alignments, we
detected RIP indices within the parameters that are
indicative of RIP in seven alignments. In addition, all
seven alignments have high transition/transversion
ratios, as is expected in sequences that are subject to
RIP.
Finally, screening of the genome sequences of Tr, Ta
and Tv identified orthologues of all genes required for
RIP in N. crassa (Table 5).
Table 3 The major classes of transposable elements found in the Trichoderma genomes
T. atroviridae T. reesei T. virens
Class Copy number Total length (bp) Copy number Total length (bp) Copy number Total length (bp)
DNA 372 39,899 446 50,448 370 52,358
LTR 533 64,534 559 76,482 541 67,484
Helitrons 40 9,235 45 9,962 34 8,547
LINE 561 65,202 530 54,928 349 59,414
Totala 178,870 (0.49%) 191,820 (0.57%) 187,803 (0.48%)
aTotal in base pairs and percentage of genome of transposable elements found in the genomes. LINE, long interspersed nuclear element; LTR, long terminal
repeat.
Table 4 Repeat-induced point mutation ratios for four of
the most abundant transposable element families in the
three Trichoderma species
Sequence TA/AT ratio CT+AT/AC+GT ratio RIPa
T. atroviride 0.70 1.35
LTR Copia 0.42 1.50
LTR Gypsy 0.97 1.21
LINE R1 1.86 1.67
LINE Tad1 0.82 1.32
T. reesei 0.71 1.28
LTR Copia 1.04 1.31
LTR Gypsy 1.01 1.28
LINE R1 0.99 2.40
LINE Tad1 0.33 1.30
T. virens 0.71 1.33
LTR Copia 0.77 1.48
LTR Gypsy 0.95 1.16
LINE R1 0.75 2.14
LINE Tad1 1.33 0.99 *
aThe asterisk indicates the family Tad1 from T. virens in which the RIP ratios
fall within values that are typically associated with RIP. LINE, long interspersed
nuclear element; LTR, long terminal repeat; RIP, repeat-induced point
mutation; TE, transposable element.
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Paralogous gene expansion in T. atroviride and T. virens
We used Marcov cluster algorithm (MCL) analysis [24]
and included ten additional ascomycete genomes pre-
sent in the Joint Genome Institute (JGI) genome data-
base (including Eurotiomycetes, Sordariomycetes and
Dothidiomycetes) to identify paralogous gene families
that have become expanded either in all three Tricho-
derma species or only in the two mycoparasitic Tricho-
derma species. Forty-six such families were identified
for all three species, of which 26 were expanded only in
Ta and Tv. The largest paralogous expansions in all
three Trichoderma species have occurred with genes
encoding Zn(2)Cys(6) transcription factors, solute trans-
porters of the major facilitator superfamily, short chain
alcohol dehydrogenases, S8 peptidases and proteins
bearing ankyrin domains (Table 6). The most expanded
protein sets, however, were those that were considerably
smaller in Tr (P < 0.05). These included ankyrin pro-
teins with CCHC zinc finger domains, proteins with
WD40, heteroincompatibility (HET) and NACHT
domains, NAD-dependent epimerases, and sugar
transporters.
Genes with possible relevance for mycoparasitism are
expanded in Trichoderma
Mycoparasitism depends on a combination of events
that include lysis of the prey’s cell walls [3,4,7]. The
necessity to degrade the carbohydrate armor of the
prey’s hyphae is reflected in an abundance of chitinolytic
enzymes (composing most of the CAZy (Carbohydrate-
Active enZYmes database) glycoside hydrolase (GH)
family GH18 fungal proteins along with more rare
endo-b-N-acetylglucosaminidases) and b-1,3-glucanases
(families GH17, GH55, GH64, and GH81) in
Trichoderma relative to other fungi. Family GH18, con-
taining enzymes involved in chitin degradation, is also
strongly expanded in Trichoderma, but particularly in
Tv and Ta, which contain the highest number of chiti-
nolytic enzymes of all described fungi (Table 7). Chitin
is a substantial component of fungal cell walls and chiti-
nases are therefore an integral part of the mycoparasitic
attack [3,25]. It is conspicuous that not only was the
number of chitinolytic enzymes elevated but that many
of these chitinases contain carbohydrate binding
domains (CBMs). Mycoparasitic Trichoderma species
are particularly rich in subgroup B chitinases that con-
tain CBM1 modules, historically described as cellulose
binding modules, but binding to chitin has also been
demonstrated [26]. Tv and Ta each have a total of five
CBM1-containing GH18 enzymes. Subgroup C chiti-
nases possess CBM18 (chitin-binding) and CBM50 mod-
ules (also known as LysM modules; described as
peptidoglycan- and chitin-binding modules). Interest-
ingly, CBM50 modules in Trichoderma are found not
only in chitinases but also frequently as multiple copies
in proteins containing a signal peptide, but with no
identifiable hydrolase domain. In most cases these genes
can be found adjacent to chitinases in the genome.
Together with the expanded presence of chitinases,
the number of GH75 chitosanases is also significantly
expanded in all three analyzed Trichoderma species. As
with plant pathogenic fungi [27,28], we have also
observed an expansion of plant cell wall degrading
enzyme gene families. A full account of all the carbohy-
drate active enzymes is presented in Tables S3 to S8 in
Additional file 1. Additional details about the Tricho-
derma CAZome (the genome-wide inventory of CAZy)
are given in Chapter 1 of Additional file 2.
Table 5 Presence of genes in Trichoderma known to be required in N. crassa for repeat-induced point mutation
N. crassa proteina Accession numbera Functiona Trichoderma orthologue (ID number)
T. atroviride T. virens T. reesei
RIP
RID XP_959047.1 Putative DMT, essential for RIP and for MIP
Dim-5 XP_957479.2 Histone 3-K9 HMT essential for RIP; RdRP 152017 55211 515216
Quelling
QDE-1 XP_959047.1 RdRP, essential for quelling 361 64774 67742
QDE-2 XP_960365.2 Argonaute-like protein, essential for quelling 79413 20883 49832
QDE-3 XP_964030.2 RecQ helicase, essential for quelling 91316 30057 102458
DCL1 XP_961898.1 Dicer-like protein, involved in quelling 20162 20212 69494
DCL2 XP_963538.2 Dicer-like protein, involved in quelling 318 47151 79823
QIP CAP68960.1 Putative exonuclease protein, involved in quelling 14588 41043 57424
MSUD
SAD-1 XP_964248.2 RdRP essential for MSUD 465 28428 103470
SAD-2 XP_961084.1 Essential for MSUD No No No
aN. crassa gene information and abbreviations taken from [36]. DMT, cytosine DNA methyltransferase; HMT, histone methyltransferase; MIP, methylation induced
premeotically; MSUD, meiotic silencing of unpaired DNA; RdRP, RNA-dependent RNA-polymerase.
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Another class of genes of possible relevance to myco-
parasitism are those involved in the formation of sec-
ondary metabolites (Chapter 2 of Additional file 2).
With respect to these, the three Trichoderma species
contained a varying assortment of non-ribosomal
peptide synthetases (NRPS) and polyketide synthases
(PKS) (Table 8; see also Tables S9 and S10 in Additional
file 1). While Tr (10 NRPS, 11 PKS and 2 NRPS/PKS
fusion genes [8]) ranked at the lower end when com-
pared to other ascomycetes, Tv exhibited the highest
Table 6 Major paralogous gene expansions in Trichoderma
PFAM domain T.
reesei
T.
virens
T.
atroviride
Other
fungia
Unknown protein with ankyrin (PF00023), CCHC zinc finger (PF00098; C-X2-C-X4-H-X4-C) and purine
nucleoside phosphorylase domain (01048)
19 38 45 4
Zn(II)Cys6 transcription factor (00172) cluster 1-5 20 43 42 5,1
Peptidase S8 subtilisin cluster 1-4 10 33 36 9,6
Unknown protein with WD40, NACHT and HET domain 13 38 35 3,4
Short chain alcohol dehydrogenase (PF00106) cluster 1 and 2 20 32 34 4,7
Unknown protein family 1-4 12 25 28 5
NAD-dependent epimerase (PFAM 01370) 10 21 23 5,8
Isoflavon reductase, plus PAPA-1 (INO80 complex subunit B), epimerase and Nmr1 domain 9 18 19 6
Ankyrin domain protein 10 17 19 8
Sugar transporters 11 24 18 10,8
GH18 chitinases 6 11 16 2
Protein kinase (00069) plus TPR domain 2 24 15 4,7
Unknown major facilitator subfamily (PF07690) domain 9 15 15 5,5
F-box domain protein 7 10 11 1,7
Ankyrin domain protein with protein kinase domain 6 8 11 2,7
Amidase 4 11 11 2,8
Epoxide hydrolase (PF06441) plus AB hydrolase_1 (PF00561) 5 14 11 3,2
FAD_binding_4, plus HET and berberine bridge enzymes (08031) domain 5 13 11 6,1
FMN oxidoreductases 2 8 10 2,5
Unknown protein with DUF84 (NTPase) and NmrA domain 5 19 10 3,7
Protein with GST_N and GST_C domains 6 12 10 4,6
Class II hydrophobins 6 8 9 1,1
Proteins with LysM binding domains 6 7 9 1,2
Unknown protein family with NmrA domain 2 11 8 0,2
Pro_CA 5 9 8 1,3
WD40 domain protein 5 11 8 2,2
C2H2 transcription factors 1 5 7 1,4
GFO_IDH_MocA (01408 and 02894) oxidoreductase 3 9 7 1,5
Protein kinase (00069) 4 6 6 0,7
Nonribosomal peptide synthase 3 4 5 1
SSCP ceratoplatanin-family 3 4 5 1
GH75 chitosanase 3 5 5 1,1
SNF2, DEAD box helicase 3 5 5 1,3
Nitrilase 3 6 5 2,2
GH65 trehalose or maltose phosphorylase (PFAM 03632) 4 4 4 0,8
AAA-family ATPase (PF00004) 4 3 4 1
Pyridoxal phosphate dependent decarboxylase (00282) 2 3 4 1,2
Unknown protein 3 4 4 1,3
aResults are from MCL analysis of the three Trichoderma species (Tr, Ta, Tv) and mean values from ten other ascomycetes whose genomes are present in the JGI
database [63]. Eurotiomycetes: Aspergillus carbonarius, Aspergillus niger. Sordariomycetes: Thielavia terrerstris, Chaetomium globosum, Cryphonectria parasitica,
Neurospora discreta, Neurospora tetrasperma. Dothidiomycetes: Mycospherella graminicola, Mycospherella fijiensis, Cochliobolus heterostrophus. The number of genes
present in the “other fungi” is averaged. Data were selected from a total of 28,919 clusters, average cluster number 5.8 (standard deviation 15.73). PFAM
categories printed in bold specify those that are significantly (P < 0.05) expanded in all three Trichoderma species; numbers in bold and italics specify genes that
are significantly more abundant in Ta and Tv versus Tr (P < 0.05). GH, glycosyl hydrolase family; GST, glutahionine-S transferase; SSCP, small secreted cystein-rich
protein.
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number (50) of PKS, NRPS and PKS-NRPS fusion genes,
mainly due to the abundance of NRPS genes (28, twice
as much as in other fungi). A phylogenetic analysis
showed that this was due to recent duplications of genes
encoding cyclodipeptide synthases, cyclosporin/enniatin
synthase-like proteins, and NRPS-hybrid proteins (Fig-
ure S1 in Additional file 3). Most of the secondary
metabolite gene clusters present in Tr were also found
in Tv and Ta, but about half of the genes remaining in
the latter two are unique for the respective species, and
are localized on non-syntenic islands of the genome (see
below). Within the NRPS, all three Trichoderma species
contained two peptaibol synthases, one for short (10 to
14 amino acids) and one for long (18 to 25 amino acids)
peptaibols. The genes encoding long peptaibol synthe-
tase lack introns and produce an mRNA that is 60 to 80
kb long that encodes proteins of approximately 25,000
amino acids, the largest fungal proteins known.
Besides PKS and NRPS, Ta and Tv have further aug-
mented their antibiotic arsenal with genes for cytolytic
peptides such as aegerolysins, pore-forming cytolysins
typically present in bacteria, fungi and plants, yeast-like
killer toxins and cyanovirins (Chapter 2 of Additional
file 2). In addition, we found two high molecular weight
toxins in Ta and Tv that bear high similarity (E-value 0
for 97% coverage) to the Tc (’toxin complex’) toxins of
Photorhabdus luminescens, a bacterium that is mutualis-
tic with entomophagous nematodes [29] (Table S11 in
Additional file 1). Apart from Trichoderma, they are
Table 7 Glycosyl hydrolase families involved in chitin/chitosan and b-1,3 glucan hydrolysis that are expanded in
mycoparasitic Trichoderma species
Glycosyl hydrolase family
Chitin/chitosana ß-glucana Total ß-glucanb
Taxonomy GH18 GH75 GH17 GH55 GH64 GH81 217
Trichoderma atroviride S 29 5 5 8 3 2 18
Trichoderma virens S 36 5 4 10 3 1 18
Trichoderma reesei S 20 3 4 6 3 2 15
Pezizomycota
Nectria haematococca S 28 2 6 5 2 1 14
Fusarium graminearum S 19 1 6 3 2 1 12
Neurospora crassa S 12 1 4 6 2 1 13
Podospora anserina S 20 1 4 7 1 1 13
Magnaporthe grisea S 14 1 7 3 1 2 13
Aspergillus nidulans E 19 2 5 6 0 1 12
Aspergillus niger E 14 2 5 3 0 1 9
Penicillium chrysogenum E 9 1 5 3 2 1 11
Tuber melanosporum P 5 1 4 2 0 3 9
Other ascomycetes
Saccharomyces cerevisiae SM 2 0 4 0 0 2 6
Schizosaccharomyces pombe SS 1 0 1 0 0 1 2
Basidiomycota
Phanerochaete chrysosporium A 11 0 2 2 0 0 4
Laccaria bicolor A 10 0 4 2 0 0 6
Postia placenta A 20 0 4 6 0 0 10
aMain substrates for the respective enzyme families. bNumber of all enzymes that can act on ß-glucan as a substrate. Taxonomy abbreviations: S,
Sordariomycetes; E, Eurotiomycetes; P, Pezizomycetes; S, Saccharomycetes; SS, Schizosaccharomycetes; A, Agaricomycetes. The bold numbers indicate glycosyl
hydrolase (GH) families that have a statistically significant expansion in Trichoderma (P < 0.05) or Ta and Tv (GH18). This support was obtained only when N.
haematococca and T. melanosporum were not included in the analysis of GH18 and GH81, respectively.
Table 8 The number of polyketide synthases and non-
ribosomal peptide synthetases of Trichoderma compared
to other fungi
Fungal species PKS NRPS PKS-NRPS
NRPS-PKS
Total
Trichoderma virens 18 28 4 50
Aspergillus oryzae 26 14 4 44
Aspergillus nidulans 26 13 1 40
Cochliobolus heterostrophus 23 11 2 36
Trichoderma atroviride 18 16 1 35
Magnaporthe oryzae 20 6 8 34
Fusarium graminearum 14 19 1 34
Gibberella moniliformis 12 16 3 31
Botryotinia fuckeliana 17 10 2 29
Aspergillus fumigatus 13 13 1 27
Nectria haematococca 12 12 1 25
Trichoderma reesei 11 10 2 23
Neurospora crassa 7 3 0 10
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also present in G. zeae and Podospora anserina. Yet
there may be several more secondary metabolite genes
to be detected: Trichoderma species contain expanded
arrays of cytochrome P450 CYP4/CYP19/CYP26 subfa-
milies (Table S12 in Additional file 1), and of soluble
epoxide hydrolases that could act on the epoxides pro-
duced by the latter (Figure S2 in Additional file 3).
The Hypocrea/Trichoderma genomes also contain an
abundant arsenal of putatively secreted proteins of 300
amino acids or less that contain at least four cysteine
residues (small secreted cysteine-rich proteins (SSCPs);
Chapter 3 of Additional file 2). They contained both
unique and shared sets of SSCPs, with a higher com-
plexity in Tv and Ta than in Tr (Table S13 in Addi-
tional file 1).
Genes present in T. atroviride and T. virens but not in T.
reesei
As mentioned above, 1,273 orthologous genes were
shared between Ta and Tv but absent from Tr. When
the encoded proteins were classified according to their
PFAM domains, fungal specific Zn(2)Cys(6) transcrip-
tion factors (PF00172, PF04082) and solute transporters
(PF07690, PF00083), all of unknown function, were
most abundant (Table S14 in Additional file 1). How-
ever, the presence of several PFAM groups of oxidore-
ductases and monooxygenases, and of enzymes for AMP
activation of acids, phosphopathetheine attachment and
synthesis of isoquinoline alkaloids was also intriguing.
This suggests that Ta and Tv may contain an as yet
undiscovered reservoir of secondary metabolites that
may contribute to their success as mycoparasites.
We also annotated the 577 genes that are unique in T.
reesei: the vast majority of them (465; 80.6%) encoded
proteins of unknown function or proteins with no
homologues in other fungi. The remaining identified
112 genes exhibited no significant abundance in particu-
lar groups, except for four Zn(2)Cys(6) transcription fac-
tors, four ankyrins, four HET-domain proteins and three
WD40-domain containing proteins.
Evolution of the non-syntenic regions
A search for overrepresentation of PFAM domains and
Gene Ontology terms in the non-syntenic regions
described above revealed that all retroposon hot spot
repeat domains [30] are found in the non-syntenic
regions. In most eukaryotes, these regions are located in
subtelomeric areas that exhibit a high recombination
frequency [31]. In addition, the genes for the protein
families in Tv and Ta that were significantly more abun-
dant compared to Tr were enriched in the non-syntenic
areas (Table 9). In addition, the number of paralogous
genes was significantly increased in the non-syntenic
regions. We considered three possible explanations for
this: the non-syntenic genes were present in the last
common ancestor of all three Trichoderma species but
were then selectively and independently lost; the non-
syntenic areas arose from the core genome by duplica-
tion and divergence during evolution of the genus Tri-
choderma; and the non-syntenic genes were acquired by
horizontal transfer. To distinguish among these hypoth-
eses for their origin, we compared the sequence charac-
teristics of the genes in the non-syntenic regions to
those present in the syntenic regions in Trichoderma
and genes in other filamentous fungi. We found that the
majority (>78%) of the syntenic as well as non-syntenic
encoded proteins have their best BLAST hit to other
ascomycete fungi, indicating that the non-syntenic
regions are also of fungal origin. Also, a high number of
proteins encoded in the non-syntenic regions of Ta and
Tv have paralogs in the syntenic region. Finally, codon
usage tables and codon adaptation index analysis [32]
indicate that the non-syntenic genes exhibit a similar
codon usage (Figure S3 in Additional file 3). Taken
together, the most parsimonious explanation for the
presence of the paralogous genes in Ta and Tv is that
the non-syntenic genes arose by gene duplication within
a Trichoderma ancestor, followed by gene loss in the
three lineages, which was much stronger in Tr.
Tr, Ta and Tv each occupy very diverse phylogenetic
positions in the genus Trichoderma, as shown by a
Bayesian rpb2 tree of 110 Trichoderma taxa (Figure 2).
In order to determine which of the three species more
likely resembles the ancestral state of Trichoderma, we
performed a Bayesian phylogenetic analysis [33] using a
Table 9 Number of PFAM domains that are enriched
among paralogous genes in non-syntenic areas
T. reesei T. virens T. atroviride
Zn2Cys6 transcription factors 9 95 69
WD40 domains 1 11 14
Sugar transporters 0 18 13
Proteases 2 28 23
Cytochrome P450 7 40 15
NmrA-domains 2 19 21
Major facilitator superfamily 7 52 60
HET domains 3 26 27
Glycoside hydrolases 3 33 26
FAD-binding proteins 2 28 24
Ankyrins 4 44 37
Alcohol dehydrogenases 4 51 71
a/ß-fold hydrolases 2 26 15
ABC transporters 4 14 3
Number of genes 50 485 418
Total gene number in NS areas 92 686 1012
Boxed numbers are those that are significantly (p < 0.05) different from the
two other species when related to the genome size. PFAM, protein family; NS,
non-syntenic; HET, heteroincompatibility.
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Figure 2 Mycoparasitism is an ancient life style of Trichoderma. (a) Position of Ta, Tv and Tr within the genus Hypocrea/Trichoderma. The
positions of Tr, Tv and Ta are 4, 29 and 97, respectively - shown in bold), and a few hallmark species are given by their names. For the identities
of the other species, see the gene accession numbers (Materials and methods). (b) Bayesian phylogram based on the analysis of amino acid
sequences of 100 orthologous syntenic proteins (MCMC, 1 million generations, 10,449 characters) in Tr, Tv, Ta, Gibberella zeae and Chaetomium
globosum. Circles above nodes indicate 100% posterior probabilities and significant bootstrap coefficients. The numbers in the boxes between (a)
and (b) indicate the genome sizes and gene counts and percentage net gain regarding Ta. Photoplates show the mycoparasitic reaction after
the contact between Trichoderma species and Rhizoctonia solani. Trichoderma species are always on the left side; dashed lines indicate the
position of Trichoderma overgrowth of R. solani.
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concatenated set of 100 proteins that were encoded by
orthologous genes in syntenic areas in the three Tricho-
derma species and also G. zeae and Chaetomium globo-
sum. The result (Figure 2) shows that Ta occurs in a
well-supported basal position to Tv and Tr. These data
indicate that Ta resembles the more ancient state of
Trichoderma and that both Tv and Tr evolved later. The
lineage to Tr thus appears to have lost a significant
number of genes present in Ta and maintained in Tv.
The long genetic distance of Tr further suggests that it
was apparently evolving faster then Ta and Tv since the
time of divergence.
To test this assumption, we compared the evolution-
ary rates of the 100 orthologous and syntenic gene
families between the three Trichoderma species. The
median values of the evolutionary rates (Ks and Ka) of
Ta-Tr and Tv-Tr were all significantly higher (1.77 and
1.47, and 1.33 and 1.19, respectively) than those of Ta-
Tv (1.13 and 0.96; all P values <0.05 by the two-tailed
Wilcoxon rank sum test). This result supports the above
suggestion that Tr has been evolving faster than Ta and
Tv.
Discussion
Comparison of the genomes of two mycoparasitic and
one saprotrophic Trichoderma species revealed remark-
able differences: in contrast to the genomes of other
multicellular ascomycetes, such as aspergilli [15,17],
those of Trichoderma appear to be have the highest
level of synteny of all genomes investigated (96% for Tr
and still 78/79% for Tv and Ta, respectively, versus 68
to 75% in aspergilli), and most of the differences
between Ta and Tv versus Tr or other ascomycetes
occur in the non-syntenic areas. Nevertheless, at a mole-
cular level the three species are as distant from each
other as apes from Pices (fishes) or Aves (birds) [17],
suggesting a mechanism maintaining this high genomic
synteny. Espagne et al. [13] proposed that a discrepancy
of genome evolution between P. anserina, N. crassa and
the aspergilli and saccharomycotina yeasts is based on
the difference between heterothallic and homothallic
fungi: in heterothallics the presence of interchromoso-
mal translocation could result in chromosome breakage
during meiosis and reduced fertility, whereas homothal-
lism allows translocations to be present in both partners
and thus have fewer consequences on fertility. Since Tri-
choderma is heterothallic [34], this explanation is also
applicable to it. However, another mechanism, meiotic
silencing of unpaired DNA [35] - which has also been
proposed for P. anserina [13], and which eliminates pro-
geny in crosses involving rearranged chromosomes in
one of the partners - may not function in Trichoderma
because one of the essential genes (SAD2 [36]) is
missing.
Our data also suggest that the ancestral state of Hypo-
crea/Trichoderma was mycoparasitic. This supports an
earlier speculation [37] that the ancestors of Tricho-
derma were mycoparasites on wood-degrading basidio-
mycetes and acquired saprotrophic characteristics to
follow their prey into their substrate. Indirect evidence
for this habitat shift in Tr was also presented by Slot
and Hibbett [38], who demonstrated that Tr - after
switching to a specialization on a nitrogen-poor habitat
(decaying wood) - has acquired a nitrate reductase gene
(which was apparently lost earlier somewhere in the
Sordariomycetes lineage) by horizontal gene transfer
from basidiomycetes.
Furthermore, the three Trichoderma species have the
lowest number of transposons reported so far. This is
unusual for filamentous fungi, as most species contain
approximately 10 to 15% repetitive DNA, primarily
composed of TEs. A notable exception is Fusarium gra-
minearum [27], which, like the Trichoderma species,
contains less than 1% repetitive DNA [8]. The paucity of
repetitive DNA may be attributed to RIP, which has
been suggested to occur in Tr [8] and for which we
have here provided evidence that it also occurs in Ta
and Tv. It is likely that this process also contributes to
prevent the accumulation of repetitive elements.
The gene inventory detected in the three Trichoderma
species reveals new insights into the physiology of this fun-
gal genus: the strong expansion of genes for solute trans-
port, oxidoreduction, and ankyrins (a family of adaptor
proteins that mediate the anchoring of ion channels or
transporters in the plasma membrane [39]) could render
Trichoderma more compatible in its habitat (for example,
to successfully compete with the other saprotrophs for lim-
iting substrates). In addition, the expansion of WD40
domains acting as hubs in cellular networks [40] could aid
in more versatile metabolism or response to stimuli. These
features correlate well with a saprotrophic lifestyle that
makes use of plant biomass that has been pre-degraded by
earlier colonizers. The expansion of HET proteins (proteins
involved in vegetative incompatibility specificity) on the
other hand suggests that Trichoderma species may fre-
quently encounter related yet genetically distinct indivi-
duals. In fact, the presence of several different Trichoderma
species can be detected in a single soil sample [41]. Unfor-
tunately, vegetative incompatibility has not yet been inves-
tigated in any Trichoderma species, and based on the
current data, should be a topic of future research.
Finally, the abundance of SSCPs in Trichoderma may
be involved in rhizosphere competence: the genome of
the ectomycorrhizal basidiomycete Laccaria bicolor also
encodes a large set of SSCPs, which accumulate in the
hyphae that colonize the host root [42].
Gene expansions in Tv and Ta that do not occur in
Tr may comprise genes specific for mycoparasitism.
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As a prominent example, proteases have expanded in Ta
and Tv, supporting the hypothesis that the degradation
of proteins is a major trait of mycoparasites [43]. Like-
wise, the increase in chitinolytic enzymes and some ß-
glucanase-containing GH families is remarkable and
illustrates the importance of destruction of the prey’s
cell wall in this process. With respect to the chitinases,
the expansion of those bearing CBM50 modules was
particularly remarkable: proteins containing these mod-
ules were recently classified into several different groups
by de Jonge and Thomma [44]. Proteins that consist
solely of CBM50 modules are type-A LysM proteins,
and there is evidence for the role of these as virulence
factors in plant pathogenic fungi. The high numbers of
LysM proteins that are found in Trichoderma, however,
indicate other/additional roles for these proteins in fun-
gal biology that are not understood yet. Also, the expan-
sion of the GH75 chitosanases was intriguing: chitosan
is a partially deacetylated derivative of chitin and,
depending on the fungal species and the growth condi-
tions, in mature fungal cell walls chitin is partially dea-
cetylated. It has also been reported that fungi
deacetylate chitin as a defense mechanism [45,46]. Chit-
osan degradation may therefore be a relevant aspect of
mycoparasitism and fungal cell wall degradation that has
also not been regarded yet. Overall, the carbohydrate-
active enzyme machinery present in Trichoderma is
compatible with saprophytic behavior but, interestingly,
the set of enzymes involved in the degradation of ‘softer’
plant cell wall components, such as pectin, is reduced.
A possible plant symbiotic relationship [3] might rely on
a mycoparasitic capacity along with a reduced specificity
for pectin, minimizing the plant defense reaction.
Although the genes encoding proteins for the synth-
esis of typical fungal secondary metabolites (PKS, NRPS,
PKS-NRPS) are also abundant, they are not significantly
more expanded than in some other fungi. An exception
is Tv and its 28 NRPS genes. However, our genome ana-
lysis revealed also a high number of oxidoreductases,
cytochrome P450 oxidases, and other enzymes that
could be part of as yet unknown pathways for the synth-
esis of further secondary metabolites. In support of this,
several of these genes were found to be clustered in the
genome (data not shown), and were more abundant in
the two mycoparasitic species Ta and Tv. Together with
the expanded set of oxidoreductases, monooxygenases,
and enzymes for AMP activation of acids, phospho-
pathetheine attachment, and synthesis of isoquinoline
alkaloids in Ta and Tv, these genes may define new sec-
ondary metabolite biosynthetic routes.
Conclusions
Our comparative genome analysis of the three Tricho-
derma species now opens new opportunities for the
development of improved and research-driven strategies
to select and improve Trichoderma species as biocontrol
agents. The availability of the genome sequences pub-
lished in this study, as well as of several pathogenic
fungi and their potential host plants (for example, [47])
provides a challenging opportunity to develop a deeper
understanding of the underlying processes by which Tri-
choderma interacts with plant pathogens in the presence
of living plants within their ecosystem.
Materials and methods
Genome sequencing and assembly
The genomes of T. virens and T. atroviride each were
assembled from shotgun reads using the JGI (USA Depart-
ment of Energy) assembler Jazz (see Table S15 in Addi-
tional file 1 for summary of assembly statistics). Each
genome was annotated using the JGI Annotation pipeline,
which combines several gene prediction, annotation and
analysis tools. Genes were predicted using Fgenesh [48],
Fgenesh+ [49], and Genewise programs [50]. ESTs from
each species (Chapter 4 of Additional file 2) were clustered
and either assembled and converted into putative full-
length genes directly mapped to genomic sequence or
used to extend predicted gene models into full-length
genes by adding 5’ and/or 3’ untranslated regions to the
models. From multiple gene models predicted at each
locus, a single representative model was chosen based on
homology and EST support and used for further analysis.
Gene model characteristics and support are summarized
in Tables S16 and S17 in Additional file 1.
All predicted gene models were functionally annotated
by homology to annotated genes from a NCBI non-
redundant set and classified according to Gene Ontology
[51], eukaryotic orthologous groups (KOGs) [52], and
Kyoto Encyclopedia of Genes and Genomes (KEGG)
metabolic pathways [53]. See Tables S18 and S19 in
Additional file 1 for a summary of the functional anno-
tation. Automatically predicted genes and functions
were further refined by user community-wide manual
curation efforts using web-based tools at [54,55]. The
latest version gene set containing manually curated
genes is called GeneCatalog.
Assembly and annotation data for Tv and Ta are
available through JGI Genome Portals homepage at
[54,55]. The genome assemblies, predicted gene models,
and annotations were deposited at GenBank under pro-
ject accessions [GenBank: ABDF00000000 and
ABDG00000000], respectively. GenBank public release
of the data described in this paper should coincide with
the manuscript publication date.
Genome similarity analysis and genomic synteny
Orthologous genes, as originally defined, imply a reflec-
tion of the history of species. In recent years, many
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studies have examined the concordance between ortho-
logous gene trees and species trees in bacteria. With the
purpose of identifying all the orthologous gene pairs for
the three Trichoderma species, a best bidirectional blast
hit approach as described elsewhere [56,57] was per-
formed, using the predicted translated gene models for
each of the three species as pairwise comparison sets.
The areas of relationship known as syntenic regions or
syntenic blocks are anchored with orthologs (calculated
as mutual best hits or bi-directional best hits) between
the two genomes in question, and are built by control-
ling for the minimum number of genes, minimum den-
sity, and maximum gap (genes not from the same
genome area) compared with randomized data as
described in [56]. While this technique may cause artifi-
cial breaks, it highlights regions that are dynamic and
picking up a large number of insertions or duplications.
Orthologous and paralogous gene models were identi-
fied by first using BLAST to find all pairwise matches
between the resulting proteins from the gene models.
The pairwise matches from BLAST were then clustered
into groups of paralogs using MCL [58]. In parallel we
applied orthoMCL [59] to the same pairwise matches to
identify the proteins that were orthologous in all of the
three genomes. By subtracting all the proteins that were
identified as orthologs from the groups of paralogs and
unique genes, we were left with only the protein pro-
ducts of gene models that have expanded since the most
recent common ancestor (MRCA) of the three Tricho-
derma genomes. We then calculated the P-value under
the null hypothesis that the number of non-orthologous
genes that are non-syntenic is less than the number of
non-orthologous genes that are syntenic.
Identification of transposable elements
We scanned the Trichoderma genomes with the de novo
repeat finding program Piler [19]. Next, we searched for
sequences with similarity to known repetitive elements
from other eukaryotes with the program RepeatMasker
[21] using all eukaryotic repetitive elements in the
RepBase (version 13.09) database. After masking repeti-
tive sequences that matched the DNA sequence of
known repetitive elements, we scanned the masked gen-
ome sequences with RepeatProteinMask (a component
of the RepeatMasker application). This search located
additional degenerate repetitive sequences with similar-
ity to proteins encoded by TEs in the RepBase database.
CAZome identification and analysis
All protein models for Ta and Tv were compared
against the set of libraries of modules derived from
CAZy [60,61]. The identified proteins were subjected to
manual analysis for correction of the protein models, for
full modular annotation and for functional inference
against a library of experimentally characterized
enzymes. Comparative analysis was made by the enu-
meration of all modules identified in the three Tricho-
derma species and 14 other published fungal genomes.
Phylogenetic and evolutionary analyses
One-hundred genes were randomly selected from Ta,
Tv, Tr and C. globosum based on their property to fulfill
two requirements: they were in synteny in all four gen-
omes, and they were true orthologues (no other gene
encoding a protein with amino acid similarity >50% pre-
sent). After alignment, the concatenated 10,449 amino
acids were subjected to Bayesian analysis [33] using 1
million generations. The respective cDNA sequences
(31,347 nucleotides) were also concatenated, and Ks/Ka
ratios determined using DNASp5 [62]. The same file
was also used to determine the codon adaptation index
[32]. In addition, 80 non-syntenic genes were also
selected randomly for this purpose.
The species phylogram of Trichoderma/Hypocrea was
constructed by Bayesian analysis of partial exon nucleo-
tide sequences (824 total characters from which 332
were parsimony-informative) of the rpb2 gene (encoding
RNA polymerase B II) from 110 ex-type strains, thereby
spanning the biodiversity of the whole genus. The tree
was obtained after 5 million MCMC generations
sampled for every 100 trees, using burnin = 1200 and
applying the general time reversible model of nucleotide
substitution. The NCBI ENTREZ accession numbers
are: 1 [HQ260620]; 3 [DQ08724]; 4 [HM182969]; 5
[HM182984]; 6 [HM182965]; 7 [AF545565]; 8
[AF545517]; 16 [FJ442769]; 17 [AY391900]; 18
[FJ179608]; 19 [FJ442715]; 20 [FJ442771]; 21
[AY391945]; 22 [EU498358]; 23 [DQ834463]; 24
[FJ442725]; 25 [AF545508]; 26 [AY391919]; 27
[AF545557]; 28 [AF545542]; 29 [FJ442738]; 30
[AF545550]; 31 [AY391909]; 32 [AF545516]; 33
[AF545518]; 34 [AF545512]; 35 [AF545510]; 36
[AF545514]; 37 [AY391921]; 38 [AF545513]; 39
[AY391954]; 40 [AY391944]; 41 [AF545534]; 42
[AY391899]; 43 [AY391907]; 44 [AF545511]; 45
[AY391929]; 46 [AF545540]; 47 [AY391958]; 48
[AY391924]; 49 [AF545515]; 50 [AY391957]; 51
[AF545551]; 52 [AF545522]; 53 [FJ442714]; 54
[AF545509]; 55 [AY391959]; 56 [DQ087239]; 57
[AF545553]; 58 [AF545545]; 59 [DQ835518]; 60
[DQ835521]; 61 [DQ835462]; 62 [DQ835465]; 63
[DQ835522]; 64 [AF545560]; 65 [DQ835517]; 66
[DQ345348]; 67 [AF545520]; 68 [DQ835455]; 69
[AF545562]; 70 [AF545563]; 71 [DQ835453]; 72
[FJ179617]; 73 [DQ859031]; 74 [EU341809]; 75
[FJ179614]; 76 [DQ087238]; 77 [AF545564]; 78
[FJ179601]; 79 [FJ179606]; 80 [FJ179612]; 81 [FJ179616];
82 [EU264004]; 83 [FJ150783]; 84 [FJ150767]; 85
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[FJ150786]; 86 [EU883559]; 87 [FJ150785]; 88
[EU248602]; 89 [EU241505]; 90 [FJ442762]; 91
[FJ442741]; 92 [FJ442783]; 93 [EU341805]; 94
[FJ442723]; 95 [FJ442772]; 96 [EU2415023]; 97
[EU341801]; 98 [EU248600]; 99 [EU341808]; 100
[EU3418033]; 101 [EU2485942]; 102 [AF545519]; 103
[EU248603]; 104 [EU248607]; 105 [EU341806]; 106
[DQ086150]; 107 [DQ834460]; 108 [EU711362]; 109
[EU883557]; 110 [FJ150790].
Additional material
Additional file 1: Comparative properties and gene inventory of T.
reesei, T. virens and T. atroviride. This file contains additional
information on genomic properties and selected gene families from the
three Trichoderma species comprising 19 tables. Table S1 summarizes the
satellite sequences identified in the Trichoderma genomes and four other
fungal genomes. Table S2 summarizes manually curated sequence
alignments of transposable element families from the Trichoderma
genomes. Table S3 lists the total number of CAZy families in Trichoderma
and other fungi. Table S4 lists the glycoside hydrolase (GH) families in
Trichoderma and other fungi. Table S5 lists the glycosyltransferase (GT)
families in Trichoderma and other fungi. Table S6 lists the polysaccharide
lyase (PL) families in Trichoderma and other fungi. Table S7 lists the
carbohydrate esterase (CE) families in Trichoderma and other fungi. Table
S8 lists the carbohydrate-binding module (CBM) families in Trichoderma
and other fungi. Table S9 lists the NRPS, PKS and NRPS-PKS proteins in T.
atroviride. Table S10 lists NRPS, PKS and NRPS-PKS proteins in T. virens.
Table S11 lists the putative insecticidal toxins in Trichoderma. Table S12
lists the cytochrome P450 CYP4/CYP19/CYP26 class E proteins in
Trichoderma. Table S13 lists the small-cysteine rich secreted protein from
Trichoderma spp. Table S14 lists the most abundant PFAM domains in
those genes that are unique to T. atroviride and T. virens and not present
in T. reesei. Table S15 surveys the assembly statistics. Table S16 provides
gene model support. Table S17 summarizes gene model statistics. Table
S18 provides numbers of genes with functional annotation according to
KOG, Gene Ontology, and KEGG classifications. Table S19 lists the largest
KOG families responsible for metabolism.
Additional file 2: Additional information on selected gene groups
of Trichoderma, methods used for genome sequencing, and
legends for the figures in Additional file 3. Chapter 1: Carbohydrate-
Active enzymes (CAZymes). Chapter 2: Aegerolysins and other toxins.
Chapter 3: Small secreted cysteine rich proteins (SSCPs). Chapter 4: EST
sequencing and analysis. Chapter 5: Legends to figures.
Additional file 3: Figures that illustrate selected aspects of the main
text. Figure S1 provides a phylogeny of Trichoderma NPRSs. Figure S2
compares the numbers of epoxide hydrolase genes in Trichoderma with
that in other fungi. Figure S3 compares the codon usage in genes from
syntenic and nonsyntenic regions of the genomes of Trichoderma reesei,
T. atroviride and T. virens.
Abbreviations
CAZy: Carbohydrate-Active enZYmes; CBM: carbohydrate binding module;
EST: expressed sequence tag; GH: glycosyl hydrolase; HET:
heteroincompatibility; KEGG: Kyoto Encyclopedia of Genes and Genomes;
KOG: clusters of eukaryotic orthologous groups; NRPS: non-ribosomal
peptide synthase; PKS: polyketide synthase; RIP: repeat-induced point
mutation; SSCP: small secreted cysteine-rich protein; Ta: Trichoderma
atroviride; TE: transposable element; Tr: Trichoderma reesei; Tv: Trichoderma
virens.
Acknowledgements
Genome sequencing and analysis was conducted by the US Department of
Energy Joint Genome Institute and supported by the Office of Science of
the US Department of Energy under contract number DE-AC02-05CH11231.
MGC-B, EYG-R, MH-O, and EEU-R are indebted to Conacyt for doctoral
fellowships. SLC and FC was supported by the Infrastructures en Biologie
Santé et Agronomie (IBISA). EM and RH work was supported by the grants
Junta de Castilla y León GR67, MICINN AGL2008-0512/AGR and AGL2009-
13431-C02. The work of ISD, VS-S, LA, BS, BM, SZ, MS, and CPK was
supported by the Austrian Science Foundation (grants FWF P17895-B06,
P20559, T390, P18109-B12, P-19421, V139B20 and P-19340). The work of PMC
and BH was supported by project number AANR-07-BIOE-006 from the
French national program PNRB. MF was the recipient of a postdoctoral
contract Ramón y Cajal from the Spanish Ministry of Science and Innovation
(MCINN: RYC-2004-003005). SZ acknowledges support from the Vienna
Science and Technology Fund (WWTF LS09-036).
Author details
1Area Gene Technology and Applied Biochemistry, Institute of Chemical
Engineering Vienna University of Technology, Getreidemarkt 9, 1060 Vienna,
Austria. 2Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav
Campus Guanajuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-León,
36821 Irapuato, Mexico. 3Broad Institute of MIT and Harvard, 301 Binney St,
Cambridge, MA 02142, USA. 4Centro Hispanoluso de Investigaciones Agrarias
(CIALE), Department of Microbiology and Genetics, University of Salamanca,
Calle Del Duero, 12, Villamayor 37185, Spain. 5División de Biología Molecular,
Instituto Potosino de Investigación Científica y Tecnológica, Camino a la
Presa San José, No. 2055, Colonia Lomas 4a Sección, San Luis Potosí, SLP.,
78216, México. 6Department of Biology, Technion - Israel Institute of
Technology, Neve Shaanan Campus, Technion City, Haifa, 32000, Israel.
7Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research
Centre, Trombay, Mumbai 400085, India. 8Department of Microbiology,
Faculty of Science and Informatics, University of Szeged, Közép fasor 52,
Szeged, H-6726, Hungary. 9DOE Joint Genome Institute, 2800 Mitchell Drive,
Walnut Creek, CA 94598, USA. 10School of Biological Sciences, University of
Missouri- Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA.
11Institut de Biologie de l’École normale supérieure (IBENS), Institut National
de la Santé et de la Recherche Médicale U1024, Centre National de la
Recherche Scientifique UMR8197, 46, rue d’Ulm, Paris 75005, France.
12Chemistry and Biomolecular Sciences, Macquarie University, Research Park
Drive Building F7B, North Ryde, Sydney, NSW 2109, Australia. 13TU Berlin,
Institut für Chemie, FG Biochemie und Molekulare Biologie OE2, Franklinstr.
29, 10587 Berlin, Germany. 14Department of Plant Pathology and
Microbiology Building 0444, Nagle Street, Texas A&M University College
Station, TX 77843, USA. 15Department of Biochemical Engineering, Faculty of
Science and Technology, University of Debrecen, Egyetem tér 1, Debrecen,
H-4010, Hungary. 16Instituto de Agroquímica y Tecnología de Alimentos,
Consejo Superior de Investigaciones Científicas, Apartado de Correos 73,
Burjassot (Valencia) E-46100, Spain. 17Architecture et Fonction des
Macromolécules Biologiques, UMR6098, CNRS, Université de la Méditerranée,
Case 932, 163 Avenue de Luminy, 13288 Marseille 13288, France.
18Department of Biotechnology, Chemistry and Environmental Engineering,
Aalborg University, Lautrupvang 15, DK-2750 Ballerup, Denmark.
19Biotechnology Department, IFP Energies nouvelles, 1-4 avenue de Bois
Préau, Rueil-Malmaison, 92852, France. 20Institute of Sciences of Food
Production (ISPA), National Research Council (CNR), Via Amendola 122/O,
70126 Bari, Italy. 21Wageningen University, Systems and Synthetic Biology,
Fungal Systems Biology Group, Dreijenplein 10, 6703 HB Wageningen, The
Netherlands. 22Chemical and Biological Process Development Group, Pacific
Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352,
USA.
Authors’ contributions
CPK, IVG, BH, EM, SEB, CMK, and AHE contributed equally to this work as
senior authors. AA, JC, MM, AS, and IVG performed global annotation and
analysis, MZ and HS did the assembly, OC and CH finished the assembly,
and EL and SL performed the genome and EST sequencing. SEB, AH-E, CMK
and CPK designed the study, and coordinated and supervised the analysis;
CPK drafted and submitted the paper. All other authors contributed research
(annotations and/or analyses). All authors read and approved the final
manuscript.
Received: 31 December 2010 Revised: 28 March 2011
Accepted: 18 April 2011 Published: 18 April 2011
Kubicek et al. Genome Biology 2011, 12:R40
http://genomebiology.com/2011/12/4/R40
Page 13 of 15

References
1. Taylor TN, Hass H, Kerp H, Krings M, Hanlin RT: Perithecial ascomycetes
from the 400 million year old Rhynie chert: an example of ancestral
polymorphism. Mycologia 2005, 97:269-285.
2. Vincent C, Goettel MS, Lazarovits G: Biological Control: A Global Perspective:
Case Studies from Around the World Wallingford, UK: CAB International; 2007.
3. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M: Trichoderma species-
opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2004, 2:43-56.
4. Howell CR: Mechanisms employed by Trichoderma species in the
biological control of plant diseases: the history and evolution of current
concepts. Plant Disease 2003, 87:4-10.
5. Harman GE: Overview of mechanisms and uses of Trichoderma spp.
Phytopathology 2006, 96:190-194.
6. Shoresh M, Harman GE, Mastouri F: Induced systemic resistance and plant
responses to fungal biocontrol agents. Annu Rev Phytopathol 2010,
48:21-43.
7. Lorito M, Woo SL, Harman GE, Monte E: Translational research on
Trichoderma: from ‘omics to the field. Annu Rev Phytopathol 2010,
48:395-417.
8. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE,
Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EG, Grigoriev IV,
Harris P, Jackson M, Kubicek CP, Han CS, Ho I, Larrondo LF, de Leon AL,
Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B,
Salamov AA, Schmoll M, Terry A, Thayer N, Westerholm-Parvinen A, et al:
Genome sequencing and analysis of the biomass-degrading fungus
Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 2008, 26:553-560.
9. Druzhinina IS, Kopchinskiy A, Kubicek CP: The first one hundred
Trichoderma species characterized by molecular data. Mycoscience 2006,
47:55-64.
10. Chaverri P, Samuels GJ, Stewart EL: Hypocrea virens sp. nov., the
teleomorph of Trichoderma virens. Mycologia 2001, 93:1113-1124.
11. Dodd SL, Lieckfeldt E, Samuels GJ: Hypocrea atroviridis sp. nov., the
teleomorph of Trichoderma atroviride. Mycologia 2003, 95:27-40.
12. Druzhinina IS, Kubicek CP, Komoń-Zelazowska M, Mulaw TB, Bissett J: The
Trichoderma harzianum demon: complex speciation history resulting in
coexistence of hypothetical biological species, recent agamospecies and
numerous relict lineages. BMC Evol Biol 2010, 10:94.
13. Espagne E, Lespinet O, Malagnac F, Da Silva C, Jaillon O, Porcel BM,
Couloux A, Aury JM, Ségurens B, Poulain J, Anthouard V, Grossetete S,
Khalili H, Coppin E, Déquard-Chablat M, Picard M, Contamine V, Arnaise S,
Bourdais A, Berteaux-Lecellier V, Gautheret D, de Vries RP, Battaglia E,
Coutinho PM, Danchin EG, Henrissat B, Khoury RE, Sainsard-Chanet A,
Boivin A, Pinan-Lucarré B, et al: The genome sequence of the model
ascomycete fungus Podospora anserina. Genome Biol 2008, 9:R77.
14. Fischer G, Rocha EP, Brunet F, Vergassola M, Dujon B: Highly variable rates
of genome rearrangements between hemiascomycetous yeast lineages.
PLoS Genet 2006, 2:e32.
15. Galagan JE, Calvo SE, Cuomo C, Ma LJ, Wortman JR, Batzoglou S, Lee SI,
Baştürkmen M, Spevak CC, Clutterbuck J, Kapitonov V, Jurka J,
Scazzocchio C, Farman M, Butler J, Purcell S, Harris S, Braus GH, Draht O,
Busch S, D’Enfert C, Bouchier C, Goldman GH, Bell-Pedersen D, Griffiths-
Jones S, Doonan JH, Yu J, Vienken K, Pain A, Freitag M, et al: Sequencing
of Aspergillus nidulans and comparative analysis with A. fumigatus and A.
oryzae. Nature 2005, 438:1105-1115.
16. Seoighe C, Federspiel N, Jones T, Hansen N, Bivolarovic V, Surzycki R,
Tamse R, Komp C, Huizar L, Davis RW, Scherer S, Tait E, Shaw DJ, Harris D,
Murphy L, Oliver K, Taylor K, Rajandream MA, Barrell BG, Wolfe KH:
Prevalence of small inversions in yeast gene order evolution. Proc Natl
Acad Sci USA 2000, 97:14433-14437.
17. Fedorova ND, Khaldi N, Joardar VS, Maiti R, Amedeo P, Anderson MJ,
Crabtree J, Silva JC, Badger JH, Albarraq A, Angiuoli S, Bussey H, Bowyer P,
Cotty PJ, Dyer PS, Egan A, Galens K, Fraser-Liggett CM, Haas BJ, Inman JM,
Kent R, Lemieux S, Malavazi I, Orvis J, Roemer T, Ronning CM, Sundaram JP,
Sutton G, Turner G, Venter JC, et al: Genomic islands in the pathogenic
filamentous fungus Aspergillus fumigatus. PLoS Genet 2008, 4:e1000046.
18. Nadeau J, Taylor B: Lengths of chromosomal segments conserved since
divergence of man and mouse. Proc Natl Acad Sci USA 1984, 81:814-818.
19. Edgar RC, Myers EW: PILER: identification and classification of genomic
repeats. Bioinformatics 2005, 21(Suppl 1):i152-i158.
20. Benson G: Tandem repeats finder: a program to analyze DNA sequences.
Nucleic Acids Res 1999, 27:573-580.
21. RepeatMasker Open-3.0.. [http://www.repeatmasker.org].
22. Margolin BS, Garrett-Engele PW, Stevens JN, Fritz DY, Garrett-Engele C,
Metzenberg RL, Selker EU: A methylated Neurospora 5S rRNA pseudogene
contains a transposable element inactivated by repeat-induced point
mutation. Genetics 1998, 149:1787-1797.
23. Selker EU, Tountas NA, Cross SH, Margolin BS, Murphy JG, Bird AP,
Freitag M: The methylated component of the Neurospora crassa genome.
Nature 2003, 422:893-897.
24. Enright AJ, van Dongen S, Ouzounis CA: An efficient algorithm for large-
scale detection of protein families. Nucleic Acids Res 2002, 30:1575-1584.
25. Seidl V: Chitinases of filamentous fungi: a large group of diverse proteins
with multiple physiological functions. Fungal Biol Rev 2008, 22:36-42.
26. Limon MC, Chacón MR, Mejías R, Delgado-Jarana J, Rincón AM, Codón AC,
Benítez T: Increased antifungal and chitinase specific activities of
Trichoderma harzianum CECT 2413 by addition of a cellulose binding
domain. Appl Microbiol Biotechnol 2004, 64:675-682.
27. Cuomo CA, Güldener U, Xu JR, Trail F, Turgeon BG, Di Pietro A, Walton JD,
Ma LJ, Baker SE, Rep M, Adam G, Antoniw J, Baldwin T, Calvo S, Chang YL,
Decaprio D, Gale LR, Gnerre S, Goswami RS, Hammond-Kosack K, Harris LJ,
Hilburn K, Kennell JC, Kroken S, Magnuson JK, Mannhaupt G, Mauceli E,
Mewes HW, Mitterbauer R, Muehlbauer G, et al: The Fusarium
graminearum genome reveals a link between localized polymorphism
and pathogen specialization. Science 2007, 317:1400-1402.
28. Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ,
Thon M, Kulkarni R, Xu JR, Pan H, Read ND, Lee YH, Carbone I, Brown D,
Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E,
Rehmeyer C, Li W, Harding M, Kim S, Lebrun MH, Bohnert H, Coughlan S,
Butler J, Calvo S, Ma LJ, et al: The genome sequence of the rice blast
fungus Magnaporthe grisea. Nature 2005, 434:980-986.
29. Münch A, Stingl L, Jung K, Heermann R: Photorhabdus luminescens genes
induced upon insect infection. BMC Genomics 2008, 9:229.
30. Wellinger RJ, Sen H: The DNA structures at the ends of eukaryotic
chromosomes. Eur J Cancer 1997, 33:735-749.
31. Freitas-Junior LH, Bottius E, Pirrit LA, Deitsch KW, Scheidig C, Guinet F,
Nehrbass U, Wellems TE, Scherf A: Frequent ectopic recombination of
virulence factor genes in telomeric chromosome clusters of P.
falciparum. Nature 2000, 407:1018-1022.
32. Sharp PM, Li WH: The codon adaptation index - a measure of directional
synonymous codon usage bias, and its potential applications. Nucleic
Acids Res 1987, 15:1281-1295.
33. Yang Z, Rannala B: Bayesian phylogenetic inference using DNA
sequences: a Markov Chain Monte Carlo Method. Mol Biol Evol 1997,
14:717-724.
34. Seidl V, Seibel C, Kubicek CP, Schmoll M: Sexual development in the
industrial workhorse Trichoderma reesei. Proc Natl Acad Sci USA 2009,
106:13909-13914.
35. Shiu PK, Metzenberg RL: Meiotic silencing by unpaired DNA: properties,
regulation and suppression. Genetics 2002, 161:1483-1495.
36. Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, Seiler S,
Bell-Pedersen D, Paietta J, Plesofsky N, Plamann M, Goodrich-Tanrikulu M,
Schulte U, Mannhaupt G, Nargang FE, Radford A, Selitrennikoff C,
Galagan JE, Dunlap JC, Loros JJ, Catcheside D, Inoue H, Aramayo R,
Polymenis M, Selker EU, Sachs MS, Marzluf GA, Paulsen I, Davis R, Ebbole DJ,
et al: Lessons from the genome sequence of Neurospora crassa: tracing
the path from genomic blueprint to multicellular organism. Microbiol Mol
Biol Rev 2004, 68:1-108.
37. Rossmann AY, Samuels GJ, Rogerson CT, Lowen R: Genera of
Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes).
Stud Mycol 1999, 42:1-83.
38. Slot JC, Hibbett DS: Horizontal transfer of a nitrate assimilation gene
cluster and ecological transitions in fungi: a phylogenetic study. PLoS
One 2007, 2:e1097.
39. Bennett V, Baines AJ: Spectrin and ankyrin-based pathways: metazoan
inventions for integrating cells into tissues. Physiol Rev 2001, 81:1353-1392.
40. Stirnimann CU, Petsalaki E, Russell RB, Müller CW: WD40 proteins propel
cellular networks. Trends Biochem Sci 2010, 35:565-574.
41. Migheli Q, Balmas V, Komoñ-Zelazowska M, Scherm B, Fiori S,
Kopchinskiy AG, Kubicek CP, Druzhinina IS: Soils of a Mediterranean hot
spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are
inhabited by pan-European, invasive species of Hypocrea/Trichoderma.
Environ Microbiol 2009, 11:35-46.
Kubicek et al. Genome Biology 2011, 12:R40
http://genomebiology.com/2011/12/4/R40
Page 14 of 15

42. Martin F, Aerts A, Ahrén D, Brun A, Danchin EG, Duchaussoy F, Gibon J,
Kohler A, Lindquist E, Pereda V: The genome of Laccaria bicolor provides
insights into mycorrhizal symbiosis. Nature 2008, 452:88-92.
43. Seidl V, Song L, Lindquist E, Gruber S, Koptchinskiy A, Zeilinger S,
Schmoll M, Martínez P, Sun J, Grigoriev I, Herrera-Estrella A, Baker SE,
Kubicek CP: Transcriptomic response of the mycoparasitic fungus
Trichoderma atroviride to the presence of a fungal prey. BMC Genomics
2009, 10:567.
44. de Jonge R, Thomma BP: Fungal LysM effectors: extinguishers of host
immunity? Trends Microbiol 2009, 17:151-154.
45. Baker LG, Specht CA, Donlin MJ, Lodge JK: Chitosan, the deacetylated
form of chitin, is necessary for cell wall integrity in Cryptococcus
neoformans. Eukaryotic Cell 2007, 6:855-862.
46. El Gueddari NE, Rauchhaus U, Moerschbacher BM, Deising HB:
Developmentally regulated conversion of surface-exposed chitin to
chitosan in cell walls of plant pathogenic fungi. New Phytol 2002,
156:103-111.
47. diArk: a resource for eukaryotic genome research.. [http://www.diark.org/
diark/search].
48. Salamov AA, Solovyev VV: Ab initio gene finding in Drosophila genomic
DNA. Genome Res 2000, 10:516-522.
49. Birney E, Durbin R: Using GeneWise in the Drosophila annotation
experiment. Genome Res 2000, 10:547-548.
50. Zdobnov EM, Apweiler R: InterProScan - an integration platform for the
signature-recognition methods in InterPro. Bioinformatics 2001,
17:847-848.
51. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP,
Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A,
Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene
Ontology: tool for the unification of biology The Gene Ontology
Consortium. Nat Genet 2000, 25:25-29.
52. Koonin EV, Fedorova ND, Jackson JD, Jacobs AR, Krylov DM, Makarova KS,
Mazumder R, Mekhedov SL, Nikolskaya AN, Rao BS, Rogozin IB, Smirnov S,
Sorokin AV, Sverdlov AV, Vasudevan S, Wolf YI, Yin JJ, Natale DA: A
comprehensive evolutionary classification of proteins encoded in
complete eukaryotic genomes. Genome Biol 2004, 5:R7.
53. Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M: The KEGG resource
for deciphering the genome. Nucleic Acids Res 2004, 32:D277-D280.
54. Trichoderma virens Gv29-8 v2.0.. [http://www.jgi.doe.gov/Tvirens].
55. Trichoderma atroviride v2.0[.. [http://www.jgi.doe.gov/Tatroviride].
56. Castillo-Ramirez S, Gonzalez V: Factors affecting the concordance between
orthologous gene trees and species tree in bacteria. BMC Evol Biol 2008,
8:300.
57. Moreno-Hagelsieb C, Janga SC: Operons and the effect of genome
redundancy in deciphering functional relationships using phylogenetic
profiles. Proteins 2008, 70:344-352.
58. MCL - a cluster algorithm for graphs.. [http://micans.org/mcl/].
59. Li L, Stoeckert CJ Jr, Roos DS: OrthoMCL: identification of ortholog groups
for eukaryotic genomes. Genome Res 2003, 13:2178-2189.
60. CAZY - Carbohydrate-Active EnZYmes database.. [http://www.cazy.org].
61. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B:
The carbohydrate-active EnZymes database (CAZy): an expert resource
for glycogenomics. Nucleic Acids Res 2009, 37:D233-D238.
62. Librado P, Rozas J: DnaSP v5: a software for comprehensive analysis of
DNA polymorphism data. Bioinformatics 2009, 25:1451-1452.
63. JGI genome portal.. [http://genome.jgi-psf.org].
64. Latgé JP: The cell wall: a carbohydrate armour for the fungal cell. Mol
Microbiol 2007, 66:279-290.
65. de Groot P, Brandt BW, Horiuchi H, Ram AF, de Koster CG, Klis FM:
Comprehensive genomic analysis of cell wall genes in Aspergillus
nidulans. Fungal Genet Biol 2009, 46(Suppl 1):S72-S81.
66. Lieckfeldt E, Kullnig CM, Samuels GJ, Kubicek CP: Sexually competent,
sucrose- and nitrate-assimilating strains of Hypocrea jecorina
(Trichoderma reesei, Hypocreales) from South American soils. Mycologia
2000, 92:374-384.
67. Vargas WA, Mandawe JC, Kenerley CM: Plant-derived sucrose is a key
element in the symbiotic association between Trichoderma virens and
maize plants. Plant Physiol 2009, 151:792-797.
68. Martens-Uzunova ES, Schaap PJ: Assessment of the pectin degrading
enzyme network of Aspergillus niger by functional genomics. Fungal
Genet Biol 2009, 46(Suppl 1):S170-S179.
69. Berne S, Lah L, Sepcić K: Aegerolysins: structure, function, and putative
biological role. Protein Sci 2009, 18:694-706.
70. Goodrich-Blair H, Clarke DJ: Mutualism and pathogenesis in Xenorhabdus
and Photorhabdus: two roads to the same destination. Mol Microbiol
2007, 64:260-268.
71. Hares MC, Hinchliffe SJ, Strong PC, Eleftherianos I, Dowling AJ, French-
Constant RH, Waterfield N: The Yersinia pseudotuberculosis and Yersinia
pestis toxin complex is active against cultured mammalian cells.
Microbiology 2008, 154:3503-3517.
72. Karasova D, Havlickova H, Sisak F, Rychlik I: Deletion of sodCI and spvBC in
Salmonella enterica serovar Enteritidis reduced its virulence to the
natural virulence of serovars Agona, Hadar and Infantis for mice but not
for chickens early after infection. Vet Microbiol 2009, 139:304-309.
73. McNulty C, Thompson J, Barrett B, Lord L, Andersen C, Roberts IS: The cell
surface expression of group 2 capsular polysaccharides in Escherichia
coli: the role of KpsD, RhsA and a multi-protein complex at the pole of
the cell. Mol Microbiol 2006, 59:907-922.
74. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment
search tool. J Mol Biol 1990, 215:403-410.
75. SignalP 3.0 Server.. [http://www.cbs.dtu.dk/services/SignalP/].
76. Brotman Y, Briff E, Viterbo A, Chet I: Role of swollenin, an expansin-like
protein from Trichoderma, in plant root colonization. Plant Physiol 2008,
147:779-789.
77. Max Planck Institute of Developmental Biology: Bioinformatics toolkit.
[http://toolkit.tuebingen.mpg.de/blastclust].
78. Talbot NJ: Growing into the air. Curr Biol 1997, 7:R78-R81.
79. Wösten HA, van Wetter MA, Lugones LG, van der Mei HC, Busscher HJ,
Wessels JG: How a fungus escapes the water to grow into the air. Curr
Biol 1999, 9:85-88.
80. Kubicek CP, Baker S, Gamauf C, Kenerley CM, Druzhinina IS: Purifying
selection and birth-and-death evolution in the class II hydrophobin
gene families of the ascomycete Trichoderma/Hypocrea. BMC Evol Biol
2008, 8:4.
81. Viterbo A, Chet I: TasHyd1, a new hydrophobin gene from the biocontrol
agent Trichoderma asperellum, is involved in plant root colonization. Mol
Plant Pathol 2006, 7:249-258.
82. Djonovic S, Pozo MJ, Dangott LJ, Howell CR, Kenerley CM: Sm1, a
proteinaceous elicitor secreted by the biocontrol fungus Trichoderma
virens induces plant defense responses and systemic resistance. Mol
Plant Microbe Interact 2006, 19:838-853.
83. Djonovic S, Vargas WA, Kolomiets MV, Horndeski M, Wiest A, Kenerley CM:
A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma
virens is required for induced systemic resistance in maize. Plant Physiol
2007, 145:875-889.
84. Rep M: Small proteins of plant-pathogenic fungi secreted during host
colonization. FEMS Microbiol Lett 2005, 253:19-27.
85. Mukherjee PK, Hadar R, Pardovitz-Kedmi E, Trushina N, Horwitz BA: MRSP1,
encoding a novel Trichoderma secreted protein, is negatively regulated
by MAPK. Biochem Biophys Res Commun 2006, 350:716-722.
86. Armaleo D, Gross SR: Structural studies on Neurospora RNA polymerases
and associated proteins. J Biol Chem 1985, 260:16174-16180.
87. Vogels 50x salts.. [http://www.fgsc.net/methods/vogels.html].
88. Jones JDG, Dunsmuir P, Bedbrook J: High-level expression of introduced
chimaeric genes in regenerated transformed plants. EMBO J 1985,
4:2411-2418.
89. Berrocal-Tito G, Sametz-Baron L, Eichenberg K, Horwitz BA, Herrera-
Estrella A: Rapid blue light regulation of a Trichoderma harzianum
photolyase gene. J Biol Chem 1999, 274:14288-14294.
90. Detter JC, Jett JM, Lucas SM, Dalin E, Arellano AR, Wang M, Nelson JR,
Chapman J, Lou Y, Rokhsar D, Hawkins TL, Richardson PM: Isothermal
strand displacement amplification applications for high-throughput
genomics. Genomics 2002, 80:691-698.
91. Huang X, Madan A: CAP3: A DNA Sequence Assembling Program.
Genome Res 1999, 9:868-877.
92. Papadopoulos JS, Agarwala R: COBALT: constraint-based alignment tool
for multiple protein sequences. Bioinformatics 2007, 23:1073-1079.
doi:10.1186/gb-2011-12-4-r40
Cite this article as: Kubicek et al.: Comparative genome sequence
analysis underscores mycoparasitism as the ancestral life style of
Trichoderma. Genome Biology 2011 12:R40.
Kubicek et al. Genome Biology 2011, 12:R40
http://genomebiology.com/2011/12/4/R40
Page 15 of 15