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GENOMICS AND PROTEOMICS
Generation, annotation, and analysis of ESTs from four
different Trichoderma strains grown under conditions
related to biocontrol
Juan Antonio Vizcaíno &José Redondo &
M. Belén Suárez &Rosa Elena Cardoza &
Rosa Hermosa &Francisco Javier González &
Manuel Rey &Enrique Monte
Received: 18 December 2006 / Revised: 8 February 2007 /Accepted: 8 February 2007 /Published online: 1 March 2007
#Springer-Verlag 2007
Abstract The functional genomics project “TrichoEST”
was developed focused on different taxonomic groups of
Trichoderma with biocontrol potential. Four cDNA librar-
ies were constructed, using similar growth conditions, from
four different Trichoderma strains: Trichoderma longibra-
chiatum T52, Trichoderma asperellum T53, Trichoderma
virens T59, and Trichoderma sp. T78. In this study, we
present the analysis of the 8,160 expressed sequence tags
(ESTs) generated. Each EST library was independently
assembled and 1,000–1,300 unique sequences were identi-
fied in each strain. First, we queried our collection of ESTs
against the NCBI nonredundant database using the
BLASTX algorithm. Moreover, using the Gene Ontology
hierarchy, we performed the annotation of 40.9% of the
unique sequences. Later, based on the EST abundance, we
examined the highly expressed genes in the four strains. A
hydrophobin was found as the gene expressed at the highest
level in two of the strains, but we also found that other
unique sequences similar to the HEX1, QID3, and NMT1
proteins were highly represented in at least two of the
Trichoderma strains.
Keywords Functional genomics .Biological control .
Mycoparasitism
Introduction
Because Trichoderma species are efficient antagonists of
other fungi and due to their ubiquitous distribution and
rapid substrate colonization, they have been commonly
used as biocontrol organisms for agriculture (Monte 2001),
and their enzyme systems are widely used in industry
(Viterbo et al. 2002). Multiple mechanisms are involved in
the antagonistic properties of Trichoderma. For instance, it
has been known for many years that they can produce a
wide range of antibiotic substances (Sivasithamparam and
Ghisalberti 1998) and that they parasitize other fungi, a
process that is called mycoparasitism (Benítez et al. 2004).
The EU-funded functional genomics project “Tricho-
EST”(http://www.trichoderma.org/),basedonthegener-
Appl Microbiol Biotechnol (2007) 75:853–862
DOI 10.1007/s00253-007-0885-0
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-007-0885-0) contains supplementary material,
which is available to authorized users.
J. A. Vizcaíno (*):M. B. Suárez
Centro de Investigaciones Científicas Isla de la Cartuja,
Instituto de Bioquímica Vegetal y Fotosíntesis,
CSIC/University of Seville,
41092 Sevilla, Spain
e-mail: javizca@usal.es
J. Redondo :F. J. González :M. Rey
Newbiotechnic, S. A. (NBT),
Parque Industrial de Bollullos A-49 (PIBO),
41110 Bollullos de la Mitación,
Sevilla, Spain
M. B. Suárez :R. Hermosa :E. Monte
Spanish–Portuguese Center of Agricultural Research (CIALE),
Departamento de Microbiología y Genética,
Universidad de Salamanca,
Edificio Departamental, lab 208, Plaza Doctores de la Reina s/n,
37007 Salamanca, Spain
R. E. Cardoza
Area of Microbiology. Escuela Superior y Técnica de Ingeniería
Agraria, Universidad de León,
Campus de Ponferrada. Avda. Astorga s/n,
24400 Ponferrada, Spain
ation of expressed sequence tags (ESTs), was undertaken
to identify genes and gene products with biotechnological
value from different Trichoderma species (Rey et al.
2004). Within this project, the strains Trichoderma long-
ibrachiatum T52, Trichoderma asperellum T53, Tricho-
derma virens T59, and Trichoderma sp. T78 (Hermosa et
al. 2004) were selected. Strain T78 had been previously
characterized as Trichoderma viride basedonitsITS1
region. However, when a fragment of the translation
elongation factor 1 (tef1) gene was analyzed, this strain
formed an independent clade from T. vi r i d e ,andwas
considered to be a member of a nondescribed species
(Hermosa et al. 2004). For this reason, it is named
Trichode rma sp.
In any case, the strains T. asperellum T53, T. virens T59,
and Trichoderma sp. T78 are typical biocontrol genotypes,
as they belong to the Trichoderma taxonomical sections
Trichoderma and Pachybasium (Hermosa et al. 2004), and
they are able to produce different fungal cell wall degrading
enzymes (Sanz et al. 2004). Strain T. longibrachiatum T52
belongs to the Trichoderma section Longibrachiatum,
which is not considered a source of strains involved in
biocontrol (Hermosa et al. 2004). However, it was included
in this study because it was found that it had high
antimicrobial activity (Vizcaíno et al. 2005).
Recently, we described the generation, annotation, and
analysis of 8,710 ESTs (3,478 unique sequences) from
Trichoderma harzianum CECT 2413 (Vizcaíno et al. 2006).
This strain represented the T. harzianum genotypes within
the “TrichoEST”project. Other studies involving EST
approaches have been carried out in Trichoderma species.
Most of them used Trichoderma reesei as a model and
pursued different goals. For example, the study of the
anaerobic and aerobic degradation of glucose (Chambergo
et al. 2002), expression profiling using microarray analysis
and identification of enzymes involved in biomass degra-
dation (Foreman et al. 2003), characterization of the protein
processing and secretion pathway (Diener et al. 2004), or
the detection of genes that were differentially expressed in
response to secretion stress (Arvas et al. 2006). Addition-
ally, Liu and Yang (2005), working with an unknown strain
of T. harzianum, used a single cDNA library made in
nondefined conditions related to biocontrol to characterize
this process.
In the present study, we report the overall analysis of
8,160 ESTs obtained from four different Trichoderma
strains: T. longibrachiatum T52, T asperellum T53, T.
virens T59, and Trichoderma sp. T78. The sequences were
derived from four different cDNA libraries that were made
in a similar way, by combining different growth conditions.
Globally, 3,544 unique sequences were identified and GO-
terms were assigned. In addition, the relative abundance of
ESTs provided a measure of gene expression.
Materials and methods
Fungal strains
T. longibrachiatum T52 (NBT52, NewBiotechnic S.A.,
Seville, Spain), T. asperellum T53 (IMI 20268, Internation-
al Mycological Institute, Egham, UK), T. virens T59 (NBT
T59), and Trichoderma sp. T78 (NBT T78) were used in
this study (Hermosa et al. 2004). The fungal strains were
maintained on potato dextrose agar (PDA, Difco Becton
Dickinson, Sparks, MD, USA).
cDNA libraries construction
Different sets of conditions, most of them designed to
simulate in vitro the biocontrol process, were used to build
mixed cDNA libraries which were made for the “Tricho-
EST”project. The libraries were made from Trichoderma
cultures obtained from almost identical growth conditions.
First, in all cases, Trichoderma was grown in a minimal
medium (Penttila et al. 1987) (MM: 15 g/l NaH
2
PO
4
, 5 g/l
(NH
4
)
2
SO
4
,600mg/lCaCl
2
·2H
2
O, 600 mg/l MgSO
4
·7H
2
O,
5 mg/l FeSO
4
, 2 mg/l CoCl
2
, 1.6 mg/l MnSO
4
,1.4mg/l
ZnSO
4
) containing 2% glucose as carbon source, in baffled
flasks at 25°C and 160 rpm for 2 days. Then, biomass was
harvested, rinsed twice with sterile distilled water, and
transferred to MM (Penttila et al. 1987) under at least the
following induction conditions in separate cultures: (1) 1.5%
chitin for 8 h, (2) MM buffered at pH 3.5 with HCl
containing 1.5% chitin, for 4 h, (3) MM containing 2%
glucose, in nitrogen starvation conditions (20 mg/l ammoni-
um sulfate) for 8 h. This protocol was exactly followed to
make the library L14T53 from T. asperellum T53.
However, to make the libraries L19T52 (from T. long-
ibrachiatum T52) and L20T59 (from T. vire ns T59), the
protocol indicated above was followed but an additional
common fourth condition of induction was included: MM
containing 1 g/l ammonium sulfate, 2% crude strawberry plant
cell walls and 0.5% glucose, incubated for 96 h without the
initial preculture (a one step culture). Additionally, to make the
library L21T78 (from Trichoderma sp. T78), a different fourth
condition of induction was included: MM containing 0.5%
polygalacturonic acid, 0.5% carboxymethylcellulose, 0.5%
pectin, 0.5% xylan, and 0.2% glucose, at 28°C incubated for
72 h, without the initial preculture (a one step culture).
Mycelia were harvested and total RNA was extracted as
previously reported (Vizcaíno et al. 2006). After the RNA
extraction, an equal amount of RNA from each of the
different growth conditions was mixed and mRNA was
purified using Dynabeads (Dynal, Oslo, Norway). The
cDNA libraries were constructed using the UNI-ZAP® XR
Vector System (Stratagene, La Jolla, CA, USA) following
the manufacturer’s instructions.
854 Appl Microbiol Biotechnol (2007) 75:853–862
Clone isolation, DNA sequencing, and sequence processing
These steps were performed exactly as previously reported
(Vizcaíno et al. 2006) and the 5′end of each selected clone
was sequenced. The data was managed and stored using
software specifically developed for the project, as described
previously (Vizcaíno et al. 2006). Briefly, EST sequencing
was performed and only sequences containing more than
150 bases, and having quality values greater than 20 from
the program Phred (Ewing and Green 1998) were selected.
Then, the EST sequences were cleaned using three
programs included in EMBOSS package (Rice et al.
2000): Vectorstrip, Trimseq, and Trimest. Finally, the EST
sequences were assembled into contigs using the program
CAP3 (Huang and Madan 1999) with default parameters.
Singlets and multisequence contigs resulting from this
curation and assembly process were annotated on MySQL
tables to build the “TrichoEST”database.
All unique sequences were queried against the NCBI
nonredundant (nr) database using the BLASTX algorithm
(Altschul et al. 1997) with default parameters. Redundancy
of the collections of ESTs was calculated as [1−(number of
unique sequences/number of sequenced ESTs)]×100. For
this purpose, we only considered those sequenced ESTs that
passed the quality criteria.
Assignment of GO terms
Annotations were based on the Gene Ontology (GO) terms
and hierarchical structure (Ashburner et al. 2000). The
unigene set of EST contigs and singlets were annotated
using the program Blast2GO (Conesa et al. 2005)using
the E-value < 10
−5
level as previously indicated (Vizcaíno
et al. 2006). The GO term annotations were merged and
loaded into the AmiGO browser and database (http://
www.godatabase.org/cgi-bin/amigo/go.cgi).
Accession numbers
The nucleotide sequences of the generated ESTs were
deposited in the EMBL nucleotide database and have been
assigned accession numbers from AJ904495 to AJ906295
(T. longibrachiatum T52), from AJ902299 to AJ904179
(T. asperellum T53), from AJ906296 to AJ907910 (T. v irens
T59), and finally from AJ907911 to AJ909448 (Tric hoderma
sp. T78), inclusive. They are available as electronic
supplementary material in the Files S1and S3.
Results
EST sequence determination
Different growth conditions, involving nutrient stress, or
simulated mycoparasitism, were used to build four mixed
cDNA libraries as described in “Materials and methods”.
Four different libraries were made and EST sequences were
produced (Table 1). A total of 6,835 ESTs were identified
as having high quality by Phred (Ewing and Green 1998)
(quality values greater than 20) from the initial 8,160
sequencing reactions (83.8%). Overall, the average se-
quence length was 514 nucleotides: 494 for L19T52 (size
range: 104–762), 550 for L14T53 (103–769), 484 for
L20T59 (101–740), and 534 for L21T78 (101–794).
Approximately 82.9% of the ESTs were longer than 400
nucleotides.
The number of sequenced ESTs and resulting unique
sequences was very similar in the four strains (Table 1).
The highest number of sequenced ESTs was obtained from
Trichoderma sp. T78 (2,208) and the highest number of
unique sequences was retrieved from T. asperellum T53
(1,323) (Table 1). These unique sequences are listed in the
File S1as supplementary material. The ESTs that are
contained in each contig are listed in the File S2, also as
supplementary material.
Comparison to the nonredundant database
Sequence comparison using the BLASTX algorithm against
the NCBI nonredundant (nr) database allowed the overall
identification of 2,953 (65.9%) unique sequences: 609
(57.8%) in T. longibrachiatum T52, 1,015 (76.7%) in T.
asperellum T53, 585 (57.8%) in T. vi re ns T59, and 744
(68.2%) in Trichoderma sp. T78. The results of this
BLASTX analysis showed that no clones were contaminated
with other organisms.
Table 1 Data of clustering and redundancy within the cDNA libraries
Library ID Sequenced ESTs Quality ESTs
a
Singlets Contigs Unique sequences Sequence redundancy (%)
L19T52 1,920 1,801 (93.8%) 757 297 1,054 41.5
L14T53 2,016 1,881 (93.3%) 1,106 217 1,323 29.7
L20T59 2,016 1,615 (80.1%) 776 236 1,012 37.3
L21T78 2,208 1,538 (69.7%) 905 186 1,091 29.1
Global 8,160 6,835 (83.8%) 3,544 936 4,480 34.5
a
Phred (Ewing and Green 1998) values greater than 20.
Appl Microbiol Biotechnol (2007) 75:853–862 855
Functional annotation and analysis
Unique sequences were assigned functions according to
gene ontology (GO) terms (Ashburner et al. 2000) based on
BLAST definitions using the program Blast2GO (Conesa et
al. 2005). Globally, GO categories were assigned to 1,831
of the 4,480 predicted unique sequences (40.9%): 379
unique sequences (36.0%) in T. longibrachiatum T52, 678
(51.2%) in T. asperellum T53, 348 (34.4%) in T. virens
T59, and 426 (39.0%) in Trichoderma sp. T78. Later, we
used a locally implemented AmiGO browser to examine the
representation of genes across different functional catego-
ries. Our AmiGO browser is publicly available in this URL:
http://www.trichoderma.org/cgi-bin/amigo_4strains/go.cgi.
The gene distribution in the main ontology categories in
each strain was studied and the percentages of unique
sequences with assigned GO terms that fell into these
categories were calculated. For this purpose, we considered
100% as the total number of unique sequences from each of
the libraries that possessed an assigned GO term in each of
the three organizing principles of GO (Biological Process,
Molecular Function and Cellular Component) (Ashburner
Table 2 Gene ontology (GO) functional assignments for the libraries L19T52, L14T53, L19T59, and L21T78
GO term GO ID T52 T53 T59 T78
Biological Process GO:0008150 327 (100%) 555 (100%) 302 (100%) 358 (100%)
Cellular process GO:0009987 299 (91.4%) 502 (90.4%) 273 (90.4%) 322 (89.9%)
Regulation of biological process GO:0050789 14 (4.3%) 36 (6.5%) 24 (7.9%) 21 (5.9%)
Response to stimulus GO:0050896 22 (6.7%) 34 (6.1%) 17 (5.6%) 23 (6.4%)
Physiological process GO:0007582 324 (99.0%) 548 (98.7%) 301 (99.7%) 352 (98.3%)
Regulation of physiological process GO:0050791 8 (2.4%) 34 (6.1%) 16 (5.3%) 19 (5.3%)
Metabolism GO:0008152 282 (86.2%) 462 (83.2%) 265 (87.7%) 286 (79.9%)
Biosynthesis GO:0009058 99 (30.3%) 180 (32.4%) 94 (31.1%) 122 (34.1%)
Catabolism GO:0009056 27 (8.3%) 53 (9.5%) 24 (7.9%) 26 (7.3%)
Cellular metabolism GO:0044237 253 (77.4%) 419 (75.5%) 238 (78.8%) 256 (71.5%)
Macromolecule metabolism GO:0043170 177 (54.1%) 286 (51.5%) 160 (53.0%) 179 (50.0%)
Nitrogen compound metabolism GO:0006807 31 (9.5%) 42 (7.6%) 32 (10.6%) 36 (10.1%)
Primary metabolism GO:0044238 213 (65.1%) 367 (66.1%) 206 (68.2%) 229 (64.0%)
Regulation of metabolism GO:0019222 7 (2.1%) 23 (4.1%) 11 (3.6%) 15 (4.2%)
Secondary metabolism GO:0019748 1 (0.31%) 3 (0.54%) 1 (0.33%) 2 (0.56%)
Molecular function GO:0003674 351 (100%) 617 (100%) 320 (100%) 384 (100%)
Binding activity GO:0005488 114 (32.5%) 238 (38.6%) 103 (32.2%) 155 (40.4%)
Cofactor binding GO:0048037 7 (2.0%) 10 (10.6%) 6 (1.9%) 7 (1.8%)
Ion binding GO:0043167 32 (9.1%) 42 (6.8%) 21 (6.6%) 28 (7.3%)
Nucleic acid binding GO:0003676 39 (11.1%) 84 (13.6%) 40 (12.5%) 60 (15.6%)
Nucleotide binding GO:0000166 37 (10.5%) 101 (16.4%) 38 (11.9%) 55 (14.3%)
Protein binding GO:0005515 17 (4.8%) 37 (6.0%) 17 (5.3%) 22 (5.7%)
Catalytic activity GO:0003824 214 (61.0%) 372 (60.3%) 206 (64.4%) 205 (53.4%)
Hydrolase activity GO:0016787 71 (20.2%) 118 (19.1%) 61 (19.1%) 60 (15.6%)
Lyase activity GO:0016829 17 (4.8%) 40 (6.5%) 20 (6.3%) 22 (5.7%)
Ligase activity GO:0016874 9 (2.6%) 19 (3.1%) 15 (4.7%) 23 (6.0%)
Oxidorreductase activity GO:0016491 77 (21.9%) 109 (17.7%) 69 (21.6%) 57 (14.8%)
Transferase activity GO:0016740 35 (10.0%) 84 (13.6%) 39 (12.2%) 43 (11.2%)
Transporter activity GO:0005215 65 (18.5%) 89 (14.4%) 53 (16.6%) 63 (16.4%)
Carrier activity GO:0005386 28 (8.0%) 46 (7.5%) 24 (7.5%) 29 (7.6%)
Ion transporter activity GO:0015075 30 (8.5%) 32 (5.2%) 20 (6.3%) 15 (3.9%)
Signal transducer activity GO:0004871 5 (1.4%) 8 (1.3%) 0 1 (0.26%)
Structural molecule activity GO:0005198 55 (15.7%) 84 (13.6%) 41 (12.8%) 51 (13.3%)
Enzyme regulator activity GO:0030234 3 (0.85%) 5 (0.81%) 1 (0.31%) 1 (0.26%)
Transcription regulator activity GO:0030528 7 (2.0%) 20 (3.2%) 9 (2.8%) 14 (3.6%)
Translation regulator activity GO:0045182 7 (2.0%) 21 (3.4%) 9 (2.8%) 9 (2.3%)
Cellular component GO:0005575 205 (100%) 379 (100%) 178 (100%) 266 (100%)
Extracellular region GO:0005576 5 (2.4%) 4 (1.1%) 1 (0.6%) 2 (0.75%)
Cell GO:0005623 201 (98.0%) 377 (99.5%) 177 (99.4%) 262 (98.5%)
Intracellular GO:0005622 165 (80.5%) 308 (81.3%) 138 (77.5%) 210 (78.9%)
Membrane GO:0016020 65 (31.7%) 134 (35.4%) 58 (32.6%) 83 (31.2%)
856 Appl Microbiol Biotechnol (2007) 75:853–862
et al. 2000). It must be taken into account that these
percentages do not add up to 100% because many deduced
proteins can have more than one GO assigned function.
Gene distribution was similar among the four Tricho-
derma strains (Table 2). However, some differences could
be found in some of the ontology categories: for example in
the library L21T78, the percentage of GO terms in the
categories “catalytic activity”(53.4%), “hydrolase activity”
(15.6%), and “ion transporter activity”(3.9%) was lower
than in the other libraries. In the library L19T52, something
similar was found for the category “regulation of physio-
logical process”(2.4%), but a higher percentage of GO
terms was found for “ion transporter activity”(8.5%).
Exploration of more abundantly expressed genes
The analysis of the frequency of specific ESTs that form
individual contigs (mRNA abundance) can give informa-
tion about the expression levels of particular genes under
different experimental conditions (Ebbole et al. 2004). An
analysis of the most abundant transcripts (found at least
eight times in each library) in each Trichoderma strain is
presented in Tables 3,4,5, and 6. A hydrophobin was the
most highly expressed transcript in L14T53 (T53C94; in
each case, the first three letters/numbers of the contig name
indicate the Trichoderma strain and the last ones, the contig
reference) and L21T78 (T78C171), whereas the most
abundant transcripts in the other two strains (T52C128
and T59C21) were unrelated sequences that displayed no
significant hits in the NCBI nr database. Hydrophobins are
small molecular weight proteins of fungal origin that
function in a diverse array of cellular processes for example
adhesion, sporulation, development, or pathogenesis
(Askolin et al. 2005).
As expected in this kind of study, a number of
housekeeping genes (involved in carbon metabolism,
energy production, or protein biosynthesis) were identified.
These included ubiquitin/polyubiquitin (in all the libraries),
the glyceraldehydephosphate dehydrogenase (T52C60 and
T53C157), the translation elongation factor 1α(T52C32), a
mannitol-1-phosphate-5-dehydrogenase, involved in the
fructose and mannose metabolism (T52C189), a phospho-
ketolase, involved in the pentose phosphate pathway
(T52C2), a ribosomal protein (T53C30), and the histones
H2A (T53C153) and H3 (T59C28 and T78C38). Many hits
similar to hypothetical or unknown function proteins of
fungal origin were also detected.
Without considering housekeeping genes or hypothetical
proteins, other highly expressed genes (in addition to the
hydrophobins) were present in at least two of the libraries
Table 3 The most abundantly represented genes in the library L19T52 from T. longibrachiatum T52
Contig ID EST count Annotation E-value
T52C128 35 Unknown N/A
T52C91 21 Unknown N/A
T52C95 18 HEX1 (Hypocrea jecorina) 2.00E-91
T52C228 16 Unknown N/A
T52C256 14 QID3 (H. lixii) 4.00E-22
T52C64 13 3-Methyl-2-oxobutanoate hydroxymethyltransferase
(ketopantoate hydroxymethyltransferase) (Aspergillus nidulans)
5.00E-21
T52C56 12 Hypothetical protein FG05928.1 (Gibberella zeae) 4.00E-14
T52C32 11 Translation elongation factor 1α(H. jecorina) E-101
T52C94 11 NMT1 protein homolog (G. zeae) 5.00E-41
T52C157 10 Unknown N/A
T52C189 10 Mannitol-1-phosphate 5-dehydrogenase-like protein
(Magnaporthe grisea)
2.00E-15
T52C217 10 Unknown N/A
T52C105 9 Conserved hypothetical protein (G. zeae) (contains InterPro
high-mobility group proteins HMG1 and HMG2 [IPR000135] domain)
5.00E-40
T52C2 9 Phosphoketolase, putative (Cryptococcus neoformans var. neoformans) 3.00E-65
T52C35 9 Hypothetical protein FG09906.1 (G. zeae) 1.00E-20
T52C47 9 Polyubiquitin (M. grisea) 2.00E-31
T52C60 9 Glyceraldehydephosphate dehydrogenase (Trichoderma koningii) 2.00E-80
T52C126 8 Hypothetical protein FG03028.1 (G. zeae) (contains InterPro
calycin-like [IPR011038] domain)
3.00E-35
T52C216 8 Unknown
T52C247 8 Hypothetical protein FG10442.1 (G. zeae) 1.00E-15
T52C267 8 Ubi4 ubiquitin family protein (Schizosaccharomyces pombe) 4.00E-96
T52C46 8 Hypothetical protein (Neurospora crassa) 4.00E-44
Appl Microbiol Biotechnol (2007) 75:853–862 857
and/or strains. First of all, a similar sequence to the HEX1
protein from Hypocrea jecorina (anamorph: T. reesei)
(Curach et al. 2004) was found in L19T52 (T52C95) and
L20T59 (T59C215). The HEX1 protein is the major
component of Woronin bodies, which are vital in mainte-
nance of the mycelial integrity of filamentous fungi by
plugging septal pores to prevent cytoplasmic bleeding in
the event of hyphal damage (Markham and Collinge 1987).
A similar sequence to the fungal cell wall protein QID3,
from Hypocrea lixii (anamorph: T. harzianum) (Lora et al.
1994) was found in L19T52 (T52C256) and L14T53
(T53C2). Finally, a similar sequence to a gene probably
involved in the synthesis of thiamine (the NMT1 protein)
was detected in L19T52 (T52C94) and L20T59 (T59C77).
Other highly expressed genes, found in only one of the
libraries and/or strains included similar sequences to a
Table 4 The most abundantly represented genes in the library L14T53 from T. asperellum T53
Contig ID EST count Annotation E-value
T53C94 37 Hydrophobin (H. lixii) 2.00E-31
T53C56 25 Hydrophobin I (H. jecorina) 5.00E-27
T53C55 24 Hypothetical protein FG03028.1 (G. zeae) 6.00E-28
T53C24 22 Hypothetical protein FG10224.1 (G. zeae) 3.00E-13
T53C3 18 Hypothetical protein FG02077.1 (G. zeae) (contains InterPro CFEM
domain, found in some proteins with a proposed role in fungal pathogenesis)
1.00E-20
T53C126 12 Unknown N/A
T53C78 12 Hypothetical protein FG05928.1 (G. zeae) 5.00E-12
T53C103 11 Cyclophilin, mitochondrial form (Tolypocladium inflatum) 4.00E-72
T53C110 11 Hydrophobin I (H. jecorina) 8.00E-16
T53C81 11 Unknown N/A
T53C2 10 QID3 (H. lixii) 3.00E-17
T53C11 9 Clock-controlled protein 6 (CCG-6) (N. crassa) 1.00E-12
T53C108 8 Polyubiquitin (Nicotiana tabacum) E-122
T53C153 8 Histone H2A (G. zeae) 5.00E-37
T53C157 8 Glyceraldehyde-3-phosphate dehydrogenase (H. lixii) 5.00E-96
T53C30 8 60 S ribosomal protein L10-A-like protein (M. grisea) E-118
Tab l e 5 The most abundantly represented genes in the library
L20T59 from T. virens T59
Contig
ID
EST
count
Annotation E-value
T59C21 26 Unknown N/A
T59C117 16 Unknown N/A
T59C213 15 Ubiquitin–ribosomal protein fusion
S27a (Candida albicans)
1.00E-22
T59C97 15 Hypothetical protein
FG08359.1 (G. zeae)
3.00E-38
T59C69 14 Unknown N/A
T59C54 12 Unknown N/A
T59C114 11 Subtilisin-like serine protease
PR1A (Metarhizium anisopliae
var. anisopliae)
2.00E-54
T59C28 11 Histone H3 (G. zeae) 1.00E-21
T59C115 10 Polyubiquitin (A. fumigatus) E-109
T59C20 10 Unknown N/A
T59C120 9 Hypothetical protein
FG10224.1 (G. zeae)
1.00E-09
T59C144 9 Unknown N/A
T59C25 9 Unknown N/A
T59C118 8 Class III chitinase precursor
(H. virens)
7.00E-08
T59C215 8 HEX1 (H. jecorina) 4.00E-86
T59C33 8 Unknown N/A
T59C49 8 Hydrophobin (H. jecorina) 2.00E-17
T59C77 8 NMT1 protein homolog
(N. crassa)
9.00E-35
Tab l e 6 The most abundantly represented genes in the library
L21T78 from Trichoderma sp. T78
Contig
ID
EST
count
Annotation E-value
T78C171 21 Hydrophobin I (H. jecorina) 2.00E-20
T78C19 15 Ubi4p (Saccharomyces cerevisiae) E-166
T78C37 15 Hydrophobin I (H. jecorina) 8.00E-17
T78C113 14 Predicted protein
(Neurospora crassa)
6.00E-20
T78C43 13 Hypothetical protein
FG08743.1 (G. zeae)
9.00E-18
T78C173 12 Hypothetical protein (N. crassa)
(contains InterPro pyridine
nucleotide-disulfide
oxidoreductase dimerization
region [IPR004099] domain)
4.00E-21
T78C29 9 Hypothetical protein
FG03028.1 (G. zeae)
4.00E-29
T78C73 9 Hypothetical protein
MG00777.4 (M. grisea)
6.00E-25
T78C38 8 Histone H3 (H. jecorina) 4.00E-69
T78C49 8 Unknown N/A
858 Appl Microbiol Biotechnol (2007) 75:853–862
ketopantoate hydroxymethyltransferase (T52C64), a cyclo-
philin (T53C103), the clock-controlled protein 6 from
Neurospora crassa (T53C11), a subtilisin-like serine
protease (T59C114), and a class III chitinase precursor
(T59C118).
Discussion
In this study, we investigate the genome of four different
Trichoderma strains using an EST-approach. This work is
part of the “TrichoEST”project, whose aim was to detect
genes with high biotechnological interest and/or involved in
the biological control process from a selection of strains
representing the main taxonomic groups that display
biocontrol activities within the genus Trichoderma (Rey et
al. 2004). Recently, we published a first study carried out in
T. harzianum CECT 2413 (Vizcaíno et al. 2006). In the
absence of a fully sequenced genome, the generation and
analysis of collections of ESTs is the method of choice to
extract novel sequence information from the targeted
organisms.
The growth conditions used to construct the cDNA
libraries were mainly chosen to simulate, in vitro, some
aspects of the biocontrol process occurring in the soil
environment like nutrient stress or mycoparasitic interac-
tions. Some growth conditions used in this study were
identical (for example, nitrogen starvation or chitin as sole
carbon source) but none of the cDNA libraries were made
in the same way as in the case of T. harzianum CECT 2413
(Vizcaíno et al. 2006). The information obtained in this
project provides a new way of discovering potential genes
involved in the biological control process. Overall, as a
preliminary approach, we searched in this collection of
ESTs for unique sequences encoding cell wall degrading
enzymes, which could be involved in the mycoparasitism.
For this purpose, we looked at the BLAST definitions and
found several unique sequences with putative chitinase (14
unique sequences), glucanase (7), or protease (85) activi-
ties. Similar results were found in T. harzianum CECT
2413 (Vizcaíno et al. 2006). This isoenzyme multiplicity
has been described before at the protein level by different
groups and has also been reported by our team in a study
where several isoenzymes with glucanase, protease, or
chitinase activities were detected in different Trichoderma
species, including T. asperellum T53 (Sanz et al. 2004).
BLASTX searches indicated that from 57.8% (in T.
longibrachiatum T52 and T. v ire n s T59) to 76.7% (in T.
asperellum T53) of the unique sequences were similar to at
least one entry in the NCBI nr database at the E-value < 10
−5
level. These percentages were lower than in Aspergillus
niger (83%) (Semova et al. 2006)andT. harzianum CECT
2413 (81%) (Vizcaíno et al. 2006), but they are similar or
even higher than in other similar EST studies that have been
carried out in other filamentous fungi like Conidiobolus
coronatus (58%) (Freimoser et al. 2003), Phakospora
pachyrhizi (48%) (Posada-Buitrago and Frederick 2005),
Schizophyllum commune (44.5%) (Guettler et al. 2003), or
Ustilago maydis (57–59%) (Austin et al. 2004;Nugentetal.
2004).
The study of the relative abundance of individual ESTs
that cluster into contigs can be used as a first indication of
transcript abundance. We identified a number of unique
sequences from each library, generated from eight or more
ESTs (Tables 3,4,5,and6). As expected, numerous
housekeeping genes and several hypothetical proteins were
detected. In two of the Trichoderma strains (T. asperellum
T53 and Trichoderma sp. T78), the most represented gene
(T53C94 and T78C171, respectively) was a sequence
similar to a hydrophobin from H. jecorina. A hydrophobin
was also the most abundantly represented gene in T.
harzianum CECT 2413 (Vizcaíno et al. 2006). It was also
present as one of the most abundant genes in the library
LT002 from T. reesei (Diener et al. 2004), but it could not
be found in a similar frequency table of any other
filamentous fungus. In some species like Magnaporthe
grisea (Kim et al. 2001)orMetarhizium anisopliae (St
Leger et al. 1992b) hydrophobins have been associated with
stress responses like nitrogen or carbon starvation. Recent-
ly, it has been described that the hydrophobins I and II from
T. reesei have a role in hyphal development and sporula-
tion, respectively (Askolin et al. 2005).
In addition to the hydrophobins, there were other highly
expressed genes that were found in more than one
Trichoderma strain. First of all, a similar sequence to the
major component of the Woronin bodies (the HEX1
protein) was found in T. longibrachiatum T52 (T52C95)
and T. virens T59 (T59C215). It was also present as one of
the most abundant genes in T. harzianum (Liu and Yang
2005). It is interesting to note that a similar sequence was
identified in other Tri chod erm a species (T. h amatum)
during the direct confrontation between Trichoderma
and the plant-pathogen fungus Sclerotinia sclerotiorum
(Carpenter et al. 2005). In that study, a subtractive
hybridization strategy was used to detect genes expressed
during mycoparasitism. In T. re e s ei,hex1 is highly expressed
during exponential growth (Curach et al. 2004). Additional-
ly, in M. grisea, it has been found that hex1 is essential for
efficient pathogenesis and for survival under nitrogen
starvation conditions. Curiously, hex1 was induced under
nitrogen starvation but limiting the amount of carbon source
did not elicit the same effect (Soundararajan et al. 2004).
Thus, according to these data, something similar could
happen in Tric hoderm a species.
A similar sequence to the chitin-induced fungal cell wall
protein QID3 from T. harzianum (Lora et al. 1994) was
Appl Microbiol Biotechnol (2007) 75:853–862 859
detected in T. longibrachiatum T52 (T52C256) and T.
asperellum T53 (T53CC2). Curiously, a QID3 precursor
was detected (Liu and Yang 2005)inT. harzianum as the
most represented gene. The role of QID3 is unknown but it
has been proposed that it is a cell wall associated protein
that is essential for cell–cell attachment. Thus, QID3 may
be functioning in appressorium formation as well as
pathogen recognition and attachment (Lora et al. 1995).
Finally, among the shared most expressed genes, a sequence
similar to the NMT1 protein was identified in T. lo ng -
ibrachiatum T52 (T52C94) and T. vi rens T59 (T59C77). It
was also identified as a highly expressed gene in T. harzianum
CECT 2413 (Vizcaíno et al. 2006), and it is probably
involved in the synthesis of thiamine (Morett et al. 2003).
Among the most represented genes found in only one of
the libraries, first of all, it must be highlighted that a
cyclophilin (T53C103) was identified in T. asperellum T53.
It was not only identified in the frequency tables obtained
in both EST studies carried out in T. harzianum (Liu and
Yang 2005; Vizcaíno et al. 2006), but also in other fungi
like Mycosphaerella graminicola (Keon et al. 2005).
Cyclophilins possess peptidyl-prolyl cis–trans isomerase
activity in vitro and can play roles in a great variety of
processes like protein folding and transport, RNA splicing,
and the regulation of multiprotein complexes in cells (Wang
and Heitman 2005). So far, cyclophilins have not been
extensively studied in filamentous fungi. However, it was
found that a cyclophilin (CYP1) from M. grisea acts as a
virulence determinant in rice blast (Viaud et al. 2002).
Secondly, a sequence similar to the clock controlled
protein 6 (CCG6) from N. crassa (T53C11) was detected in
the same strain. It was present as the second most abundant
gene in a study carried out in the wheat pathogen M.
graminicola (Keon et al. 2005). In N. crassa,ccg6
expression is cyclical, peaking during the late night to
early morning hours. It has been proposed that it is both
developmentally regulated and photoinducible and it could
be involved in conidiation (Bell-Pedersen et al. 1996).
It is interesting to note that two unique sequences that
could be involved in the mycoparasitism process were also
detected as highly abundant genes in T. vi rens T59:
sequences similar to a subtilisin serine protease
(T59C114) and a possible chitinase precursor (T59C118).
It has been demonstrated that this protease (PR1A)
participates in the penetration of the insect cuticle by the
entomopathogenic fungus M. anisopliae (St Leger et al.
1992a). In fact, the efficacy of this fungus as a biological
control agent can be substantially improved by over-
expression of PR1A (St Leger et al. 1996). Curiously, no
cell-wall degrading enzyme has been detected in the
frequency tables obtained in both the related to biocontrol
collections of ESTs from T. harzianum (Liu and Yang 2005;
Vizcaíno et al. 2006).
Finally, a similar sequence to a ketopantoate hydroxy-
methyl transferase (T52C64) was detected in T. long-
ibrachiatum T52, a strain that displays strong antimicrobial
activity. This enzyme is responsible for the first step in the
biosynthesis of the coenzyme A, but also of the pathway
intermediate 4′-phosphopantetheine, an essential prosthetic
group for the activity of a family of enzymes called peptide
synthetases (Kurtov et al. 1999). These large proteins are
involved in the synthesis of nonribosomal peptides, some of
them possessing antibiotic activities, and they could also be
involved in the biocontrol process.
When we performed the annotation of the sequences, the
overall percentage of assigned GO-terms (40.9%) was
lower than in T. harzianum CECT 2413 (51.1%) (Vizcaíno
et al. 2006), using the same automatic annotation method.
A similar percentage of GO-annotated unique sequences
was only achieved in T. asperellum T53 (51.2%). The
distribution of the GO terms among the Trichoderma strains
was quite similar, which is logical taking into account that
the four cDNA libraries were made in similar growth
conditions (Table 2).
When we compared the GO-term distribution in these
libraries with the similar study made in the cDNA libraries
obtained from T. harzianum CECT 2413 (Vizcaíno et al.
2006), the distribution was quite similar to the libraries
L02, L03, and L10. However, significant differences were
found between the library L06 and the rest (including those
coming from this work). The library L06 was made from
Trichoderma growing in solid media and this could be the
reason for these changes in the distribution, although other
explanations cannot be overruled (Vizcaíno et al. 2006). As
some of the growth conditions were shared among the
Trichoderma species included in the “TrichoEST”project,
and although none of the cDNA libraries made for the
present work were obtained in identical conditions to those
used in T. harzianum CECT 2413 (Vizcaíno et al. 2006), it
seems logical to find a comparable distribution of the GO
terms in all the strains.
Among the strains studied in this work, T. longibrachia-
tum T52 is the only one that belongs to the taxonomical
sect. Longibrachiatum, whose members are not considered
to be biocontrol agents (Hermosa et al. 2004). This strain is
also the only one isolated from a nonagricultural source
because it was found in a water treatment plant. In a
previous study (Vizcaíno et al. 2005), we screened the
antimicrobial activities in selected isolates representing
three Trichoderma sections. T. longibrachiatum showed
the best nonenzymatic antimicrobial profiles against bacte-
ria, yeasts, and filamentous fungi, suggesting that its
antibiotic mechanism could be more relevant than its
glucacolytic/chitinolytic mode of action. Moreover, an
isoenzyme study showed that T. longibrachiatum was able
to produce glucanases, chitinases, and proteases. However,
860 Appl Microbiol Biotechnol (2007) 75:853–862
extracellular protein extracts were not effective in the
inhibition of the hyphal growth of Botrytis cinerea (Sanz
et al. 2004). This could explain our results (both the profile
of highly represented genes and the distribution of the GO
terms in T. longibrachiatum T52 could be considered
similar to the other strains). Because Trichoderma can
integrate several biocontrol mechanisms (Howell 2003), it
seems that there are additional environmental factors giving
similar transcriptomes with different biological functions.
The relevance of the strain T. longibrachiatum T52 as a
biocontrol agent is now subject to study mainly in base to
their metabolite production with antifungal activity.
The 8,160 ESTs obtained in this study represent the
major attempt so far to define the gene set of these
Trichoderma species. Overall, they represent about 4,480
unique sequences from four different Trichoderma strains, a
fact that dramatically increases the number of identified
Trichoderma genes, reflecting also the genetic diversity
with this genus.
Acknowledgements First, the authors want to acknowledge the
financial support of the European Commission to the project
“TrichoEST”(QLK3-CT-2002-02032) and the “Fundación Ramón
Areces”. We want to recognize the work carried out by I. Chamorro,
E. Keck, J.A. de Cote, I. González, and M. Andrada for their technical
support. Authors want also to acknowledge R. Jiménez, A. Gaignard,
M. P. García-Pastor, and J. Heinrich, who helped in different
bioinformatics related tasks. Additionally, we want to thank C.
Mungall for his help in setting up the AmiGO browser. This
manuscript is dedicated to Prof. Antonio Llobell because without his
contribution, this project could not be carried out.
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