GENOMICS AND PROTEOMICS

Generation, annotation, and analysis of ESTs from fourdifferent Trichoderma strains grown under conditionsrelated 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 ofTrichoderma with biocontrol potential. Four cDNA librar-ies were constructed, using similar growth conditions, fromfour different Trichoderma strains: Trichoderma longibra-chiatum T52, Trichoderma asperellum T53, Trichodermavirens T59, and Trichoderma sp. T78. In this study, wepresent the analysis of the 8,160 expressed sequence tags

(ESTs) generated. Each EST library was independentlyassembled and 1,000–1,300 unique sequences were identi-fied in each strain. First, we queried our collection of ESTsagainst the NCBI nonredundant database using theBLASTX algorithm. Moreover, using the Gene Ontologyhierarchy, we performed the annotation of 40.9% of theunique sequences. Later, based on the EST abundance, weexamined the highly expressed genes in the four strains. Ahydrophobin was found as the gene expressed at the highestlevel in two of the strains, but we also found that otherunique sequences similar to the HEX1, QID3, and NMT1proteins were highly represented in at least two of theTrichoderma strains.

Keywords Functional genomics . Biological control .

Mycoparasitism

Introduction

Because Trichoderma species are efficient antagonists ofother fungi and due to their ubiquitous distribution andrapid substrate colonization, they have been commonlyused as biocontrol organisms for agriculture (Monte 2001),and their enzyme systems are widely used in industry(Viterbo et al. 2002). Multiple mechanisms are involved inthe antagonistic properties of Trichoderma. For instance, ithas been known for many years that they can produce awide range of antibiotic substances (Sivasithamparam andGhisalberti 1998) and that they parasitize other fungi, aprocess that is called mycoparasitism (Benítez et al. 2004).

The EU-funded functional genomics project “Tricho-EST” (http://www.trichoderma.org/), based on the gener-

Appl Microbiol Biotechnol (2007) 75:853–862DOI 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. SuárezCentro de Investigaciones Científicas Isla de la Cartuja,Instituto de Bioquímica Vegetal y Fotosíntesis,CSIC/University of Seville,41092 Sevilla, Spaine-mail: javizca@usal.es

J. Redondo : F. J. González :M. ReyNewbiotechnic, S. A. (NBT),Parque Industrial de Bollullos A-49 (PIBO),41110 Bollullos de la Mitación,Sevilla, Spain

M. B. Suárez :R. Hermosa : E. MonteSpanish–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. CardozaArea of Microbiology. Escuela Superior y Técnica de IngenieríaAgraria, Universidad de León,Campus de Ponferrada. Avda. Astorga s/n,24400 Ponferrada, Spain

ation of expressed sequence tags (ESTs), was undertakento identify genes and gene products with biotechnologicalvalue 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 etal. 2004) were selected. Strain T78 had been previouslycharacterized as Trichoderma viride based on its ITS1region. However, when a fragment of the translationelongation factor 1 (tef1) gene was analyzed, this strainformed an independent clade from T. viride, and wasconsidered to be a member of a nondescribed species(Hermosa et al. 2004). For this reason, it is namedTrichoderma 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 sectionsTrichoderma and Pachybasium (Hermosa et al. 2004), andthey are able to produce different fungal cell wall degradingenzymes (Sanz et al. 2004). Strain T. longibrachiatum T52belongs to the Trichoderma section Longibrachiatum,which is not considered a source of strains involved inbiocontrol (Hermosa et al. 2004). However, it was includedin this study because it was found that it had highantimicrobial activity (Vizcaíno et al. 2005).

Recently, we described the generation, annotation, andanalysis of 8,710 ESTs (3,478 unique sequences) fromTrichoderma harzianum CECT 2413 (Vizcaíno et al. 2006).This strain represented the T. harzianum genotypes withinthe “TrichoEST” project. Other studies involving ESTapproaches have been carried out in Trichoderma species.Most of them used Trichoderma reesei as a model andpursued different goals. For example, the study of theanaerobic and aerobic degradation of glucose (Chambergoet al. 2002), expression profiling using microarray analysisand identification of enzymes involved in biomass degra-dation (Foreman et al. 2003), characterization of the proteinprocessing and secretion pathway (Diener et al. 2004), orthe detection of genes that were differentially expressed inresponse to secretion stress (Arvas et al. 2006). Addition-ally, Liu and Yang (2005), working with an unknown strainof T. harzianum, used a single cDNA library made innondefined conditions related to biocontrol to characterizethis process.

In the present study, we report the overall analysis of8,160 ESTs obtained from four different Trichodermastrains: T. longibrachiatum T52, T asperellum T53, T.virens T59, and Trichoderma sp. T78. The sequences werederived from four different cDNA libraries that were madein 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 ofESTs 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 (NBTT59), and Trichoderma sp. T78 (NBT T78) were used inthis study (Hermosa et al. 2004). The fungal strains weremaintained on potato dextrose agar (PDA, Difco BectonDickinson, Sparks, MD, USA).

cDNA libraries construction

Different sets of conditions, most of them designed tosimulate in vitro the biocontrol process, were used to buildmixed cDNA libraries which were made for the “Tricho-EST” project. The libraries were made from Trichodermacultures obtained from almost identical growth conditions.First, in all cases, Trichoderma was grown in a minimalmedium (Penttila et al. 1987) (MM: 15 g/l NaH2PO4, 5 g/l(NH4)2SO4, 600 mg/l CaCl2·2H2O, 600 mg/l MgSO4·7H2O,5 mg/l FeSO4, 2 mg/l CoCl2, 1.6 mg/l MnSO4, 1.4 mg/lZnSO4) containing 2% glucose as carbon source, in baffledflasks at 25°C and 160 rpm for 2 days. Then, biomass washarvested, rinsed twice with sterile distilled water, andtransferred to MM (Penttila et al. 1987) under at least thefollowing induction conditions in separate cultures: (1) 1.5%chitin for 8 h, (2) MM buffered at pH 3.5 with HClcontaining 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 tomake the library L14T53 from T. asperellum T53.

However, to make the libraries L19T52 (from T. long-ibrachiatum T52) and L20T59 (from T. virens T59), theprotocol indicated above was followed but an additionalcommon fourth condition of induction was included: MMcontaining 1 g/l ammonium sulfate, 2% crude strawberry plantcell walls and 0.5% glucose, incubated for 96 h without theinitial preculture (a one step culture). Additionally, to make thelibrary L21T78 (from Trichoderma sp. T78), a different fourthcondition 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 for72 h, without the initial preculture (a one step culture).

Mycelia were harvested and total RNA was extracted aspreviously reported (Vizcaíno et al. 2006). After the RNAextraction, an equal amount of RNA from each of thedifferent growth conditions was mixed and mRNA waspurified using Dynabeads (Dynal, Oslo, Norway). ThecDNA libraries were constructed using the UNI-ZAP® XRVector System (Stratagene, La Jolla, CA, USA) followingthe 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 clonewas sequenced. The data was managed and stored usingsoftware specifically developed for the project, as describedpreviously (Vizcaíno et al. 2006). Briefly, EST sequencingwas performed and only sequences containing more than150 bases, and having quality values greater than 20 fromthe program Phred (Ewing and Green 1998) were selected.Then, the EST sequences were cleaned using threeprograms included in EMBOSS package (Rice et al.2000): Vectorstrip, Trimseq, and Trimest. Finally, the ESTsequences were assembled into contigs using the programCAP3 (Huang and Madan 1999) with default parameters.Singlets and multisequence contigs resulting from thiscuration and assembly process were annotated on MySQLtables to build the “TrichoEST” database.

All unique sequences were queried against the NCBInonredundant (nr) database using the BLASTX algorithm(Altschul et al. 1997) with default parameters. Redundancyof the collections of ESTs was calculated as [1−(number ofunique sequences/number of sequenced ESTs)]×100. Forthis purpose, we only considered those sequenced ESTs thatpassed the quality criteria.

Assignment of GO terms

Annotations were based on the Gene Ontology (GO) termsand hierarchical structure (Ashburner et al. 2000). Theunigene set of EST contigs and singlets were annotatedusing the program Blast2GO (Conesa et al. 2005) usingthe E-value < 10−5 level as previously indicated (Vizcaínoet al. 2006). The GO term annotations were merged andloaded into the AmiGO browser and database (http://www.godatabase.org/cgi-bin/amigo/go.cgi).

Accession numbers

The nucleotide sequences of the generated ESTs weredeposited in the EMBL nucleotide database and have beenassigned accession numbers from AJ904495 to AJ906295(T. longibrachiatum T52), from AJ902299 to AJ904179

(T. asperellum T53), from AJ906296 to AJ907910 (T. virensT59), and finally from AJ907911 to AJ909448 (Trichodermasp. T78), inclusive. They are available as electronicsupplementary material in the Files S1 and S3.

Results

EST sequence determination

Different growth conditions, involving nutrient stress, orsimulated mycoparasitism, were used to build four mixedcDNA libraries as described in “Materials and methods”.Four different libraries were made and EST sequences wereproduced (Table 1). A total of 6,835 ESTs were identifiedas having high quality by Phred (Ewing and Green 1998)(quality values greater than 20) from the initial 8,160sequencing reactions (83.8%). Overall, the average se-quence length was 514 nucleotides: 494 for L19T52 (sizerange: 104–762), 550 for L14T53 (103–769), 484 forL20T59 (101–740), and 534 for L21T78 (101–794).Approximately 82.9% of the ESTs were longer than 400nucleotides.

The number of sequenced ESTs and resulting uniquesequences was very similar in the four strains (Table 1).The highest number of sequenced ESTs was obtained fromTrichoderma sp. T78 (2,208) and the highest number ofunique sequences was retrieved from T. asperellum T53(1,323) (Table 1). These unique sequences are listed in theFile S1 as supplementary material. The ESTs that arecontained in each contig are listed in the File S2, also assupplementary material.

Comparison to the nonredundant database

Sequence comparison using the BLASTX algorithm againstthe NCBI nonredundant (nr) database allowed the overallidentification 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. virens T59, and 744(68.2%) in Trichoderma sp. T78. The results of thisBLASTX analysis showed that no clones were contaminatedwith other organisms.

Table 1 Data of clustering and redundancy within the cDNA libraries

Library ID Sequenced ESTs Quality ESTsa Singlets Contigs Unique sequences Sequence redundancy (%)

L19T52 1,920 1,801 (93.8%) 757 297 1,054 41.5L14T53 2,016 1,881 (93.3%) 1,106 217 1,323 29.7L20T59 2,016 1,615 (80.1%) 776 236 1,012 37.3L21T78 2,208 1,538 (69.7%) 905 186 1,091 29.1Global 8,160 6,835 (83.8%) 3,544 936 4,480 34.5

a Phred (Ewing and Green 1998) values greater than 20.

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Functional annotation and analysis

Unique sequences were assigned functions according togene ontology (GO) terms (Ashburner et al. 2000) based onBLAST definitions using the program Blast2GO (Conesa etal. 2005). Globally, GO categories were assigned to 1,831of the 4,480 predicted unique sequences (40.9%): 379unique sequences (36.0%) in T. longibrachiatum T52, 678(51.2%) in T. asperellum T53, 348 (34.4%) in T. virensT59, and 426 (39.0%) in Trichoderma sp. T78. Later, weused 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 ineach strain was studied and the percentages of uniquesequences with assigned GO terms that fell into thesecategories were calculated. For this purpose, we considered100% as the total number of unique sequences from each ofthe libraries that possessed an assigned GO term in each ofthe 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%)

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et al. 2000). It must be taken into account that thesepercentages do not add up to 100% because many deducedproteins can have more than one GO assigned function.

Gene distribution was similar among the four Tricho-derma strains (Table 2). However, some differences couldbe found in some of the ontology categories: for example inthe library L21T78, the percentage of GO terms in thecategories “catalytic activity” (53.4%), “hydrolase activity”(15.6%), and “ion transporter activity” (3.9%) was lowerthan in the other libraries. In the library L19T52, somethingsimilar was found for the category “regulation of physio-logical process” (2.4%), but a higher percentage of GOterms was found for “ion transporter activity” (8.5%).

Exploration of more abundantly expressed genes

The analysis of the frequency of specific ESTs that formindividual contigs (mRNA abundance) can give informa-tion about the expression levels of particular genes underdifferent experimental conditions (Ebbole et al. 2004). Ananalysis of the most abundant transcripts (found at leasteight times in each library) in each Trichoderma strain ispresented in Tables 3, 4, 5, and 6. A hydrophobin was themost highly expressed transcript in L14T53 (T53C94; ineach case, the first three letters/numbers of the contig name

indicate the Trichoderma strain and the last ones, the contigreference) and L21T78 (T78C171), whereas the mostabundant transcripts in the other two strains (T52C128and T59C21) were unrelated sequences that displayed nosignificant hits in the NCBI nr database. Hydrophobins aresmall molecular weight proteins of fungal origin thatfunction in a diverse array of cellular processes for exampleadhesion, sporulation, development, or pathogenesis(Askolin et al. 2005).

As expected in this kind of study, a number ofhousekeeping genes (involved in carbon metabolism,energy production, or protein biosynthesis) were identified.These included ubiquitin/polyubiquitin (in all the libraries),the glyceraldehydephosphate dehydrogenase (T52C60 andT53C157), the translation elongation factor 1α (T52C32), amannitol-1-phosphate-5-dehydrogenase, involved in thefructose and mannose metabolism (T52C189), a phospho-ketolase, involved in the pentose phosphate pathway(T52C2), a ribosomal protein (T53C30), and the histonesH2A (T53C153) and H3 (T59C28 and T78C38). Many hitssimilar to hypothetical or unknown function proteins offungal origin were also detected.

Without considering housekeeping genes or hypotheticalproteins, other highly expressed genes (in addition to thehydrophobins) 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/AT52C91 21 Unknown N/AT52C95 18 HEX1 (Hypocrea jecorina) 2.00E-91T52C228 16 Unknown N/AT52C256 14 QID3 (H. lixii) 4.00E-22T52C64 13 3-Methyl-2-oxobutanoate hydroxymethyltransferase

(ketopantoate hydroxymethyltransferase) (Aspergillus nidulans)5.00E-21

T52C56 12 Hypothetical protein FG05928.1 (Gibberella zeae) 4.00E-14T52C32 11 Translation elongation factor 1α (H. jecorina) E-101T52C94 11 NMT1 protein homolog (G. zeae) 5.00E-41T52C157 10 Unknown N/AT52C189 10 Mannitol-1-phosphate 5-dehydrogenase-like protein

(Magnaporthe grisea)2.00E-15

T52C217 10 Unknown N/AT52C105 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-65T52C35 9 Hypothetical protein FG09906.1 (G. zeae) 1.00E-20T52C47 9 Polyubiquitin (M. grisea) 2.00E-31T52C60 9 Glyceraldehydephosphate dehydrogenase (Trichoderma koningii) 2.00E-80T52C126 8 Hypothetical protein FG03028.1 (G. zeae) (contains InterPro

calycin-like [IPR011038] domain)3.00E-35

T52C216 8 UnknownT52C247 8 Hypothetical protein FG10442.1 (G. zeae) 1.00E-15T52C267 8 Ubi4 ubiquitin family protein (Schizosaccharomyces pombe) 4.00E-96T52C46 8 Hypothetical protein (Neurospora crassa) 4.00E-44

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and/or strains. First of all, a similar sequence to the HEX1protein from Hypocrea jecorina (anamorph: T. reesei)(Curach et al. 2004) was found in L19T52 (T52C95) andL20T59 (T59C215). The HEX1 protein is the majorcomponent of Woronin bodies, which are vital in mainte-

nance of the mycelial integrity of filamentous fungi byplugging septal pores to prevent cytoplasmic bleeding inthe 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 probablyinvolved 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 thelibraries 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-31T53C56 25 Hydrophobin I (H. jecorina) 5.00E-27T53C55 24 Hypothetical protein FG03028.1 (G. zeae) 6.00E-28T53C24 22 Hypothetical protein FG10224.1 (G. zeae) 3.00E-13T53C3 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/AT53C78 12 Hypothetical protein FG05928.1 (G. zeae) 5.00E-12T53C103 11 Cyclophilin, mitochondrial form (Tolypocladium inflatum) 4.00E-72T53C110 11 Hydrophobin I (H. jecorina) 8.00E-16T53C81 11 Unknown N/AT53C2 10 QID3 (H. lixii) 3.00E-17T53C11 9 Clock-controlled protein 6 (CCG-6) (N. crassa) 1.00E-12T53C108 8 Polyubiquitin (Nicotiana tabacum) E-122T53C153 8 Histone H2A (G. zeae) 5.00E-37T53C157 8 Glyceraldehyde-3-phosphate dehydrogenase (H. lixii) 5.00E-96T53C30 8 60 S ribosomal protein L10-A-like protein (M. grisea) E-118

Table 5 The most abundantly represented genes in the libraryL20T59 from T. virens T59

ContigID

ESTcount

Annotation E-value

T59C21 26 Unknown N/AT59C117 16 Unknown N/AT59C213 15 Ubiquitin–ribosomal protein fusion

S27a (Candida albicans)1.00E-22

T59C97 15 Hypothetical proteinFG08359.1 (G. zeae)

3.00E-38

T59C69 14 Unknown N/AT59C54 12 Unknown N/AT59C114 11 Subtilisin-like serine protease

PR1A (Metarhizium anisopliaevar. anisopliae)

2.00E-54

T59C28 11 Histone H3 (G. zeae) 1.00E-21T59C115 10 Polyubiquitin (A. fumigatus) E-109T59C20 10 Unknown N/AT59C120 9 Hypothetical protein

FG10224.1 (G. zeae)1.00E-09

T59C144 9 Unknown N/AT59C25 9 Unknown N/AT59C118 8 Class III chitinase precursor

(H. virens)7.00E-08

T59C215 8 HEX1 (H. jecorina) 4.00E-86T59C33 8 Unknown N/AT59C49 8 Hydrophobin (H. jecorina) 2.00E-17T59C77 8 NMT1 protein homolog

(N. crassa)9.00E-35

Table 6 The most abundantly represented genes in the libraryL21T78 from Trichoderma sp. T78

ContigID

ESTcount

Annotation E-value

T78C171 21 Hydrophobin I (H. jecorina) 2.00E-20T78C19 15 Ubi4p (Saccharomyces cerevisiae) E-166T78C37 15 Hydrophobin I (H. jecorina) 8.00E-17T78C113 14 Predicted protein

(Neurospora crassa)6.00E-20

T78C43 13 Hypothetical proteinFG08743.1 (G. zeae)

9.00E-18

T78C173 12 Hypothetical protein (N. crassa)(contains InterPro pyridinenucleotide-disulfideoxidoreductase dimerizationregion [IPR004099] domain)

4.00E-21

T78C29 9 Hypothetical proteinFG03028.1 (G. zeae)

4.00E-29

T78C73 9 Hypothetical proteinMG00777.4 (M. grisea)

6.00E-25

T78C38 8 Histone H3 (H. jecorina) 4.00E-69T78C49 8 Unknown N/A

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ketopantoate hydroxymethyltransferase (T52C64), a cyclo-philin (T53C103), the clock-controlled protein 6 fromNeurospora crassa (T53C11), a subtilisin-like serineprotease (T59C114), and a class III chitinase precursor(T59C118).

Discussion

In this study, we investigate the genome of four differentTrichoderma strains using an EST-approach. This work ispart of the “TrichoEST” project, whose aim was to detectgenes with high biotechnological interest and/or involved inthe biological control process from a selection of strainsrepresenting the main taxonomic groups that displaybiocontrol activities within the genus Trichoderma (Rey etal. 2004). Recently, we published a first study carried out inT. harzianum CECT 2413 (Vizcaíno et al. 2006). In theabsence of a fully sequenced genome, the generation andanalysis of collections of ESTs is the method of choice toextract novel sequence information from the targetedorganisms.

The growth conditions used to construct the cDNAlibraries were mainly chosen to simulate, in vitro, someaspects of the biocontrol process occurring in the soilenvironment like nutrient stress or mycoparasitic interac-tions. Some growth conditions used in this study wereidentical (for example, nitrogen starvation or chitin as solecarbon source) but none of the cDNA libraries were madein the same way as in the case of T. harzianum CECT 2413(Vizcaíno et al. 2006). The information obtained in thisproject provides a new way of discovering potential genesinvolved in the biological control process. Overall, as apreliminary approach, we searched in this collection ofESTs for unique sequences encoding cell wall degradingenzymes, which could be involved in the mycoparasitism.For this purpose, we looked at the BLAST definitions andfound several unique sequences with putative chitinase (14unique sequences), glucanase (7), or protease (85) activi-ties. Similar results were found in T. harzianum CECT2413 (Vizcaíno et al. 2006). This isoenzyme multiplicityhas been described before at the protein level by differentgroups and has also been reported by our team in a studywhere several isoenzymes with glucanase, protease, orchitinase activities were detected in different Trichodermaspecies, including T. asperellum T53 (Sanz et al. 2004).

BLASTX searches indicated that from 57.8% (in T.longibrachiatum T52 and T. virens T59) to 76.7% (in T.asperellum T53) of the unique sequences were similar to atleast one entry in the NCBI nr database at the E-value < 10−5

level. These percentages were lower than in Aspergillusniger (83%) (Semova et al. 2006) and T. harzianum CECT2413 (81%) (Vizcaíno et al. 2006), but they are similar or

even higher than in other similar EST studies that have beencarried out in other filamentous fungi like Conidioboluscoronatus (58%) (Freimoser et al. 2003), Phakosporapachyrhizi (48%) (Posada-Buitrago and Frederick 2005),Schizophyllum commune (44.5%) (Guettler et al. 2003), orUstilago maydis (57–59%) (Austin et al. 2004; Nugent et al.2004).

The study of the relative abundance of individual ESTsthat cluster into contigs can be used as a first indication oftranscript abundance. We identified a number of uniquesequences from each library, generated from eight or moreESTs (Tables 3, 4, 5, and 6). As expected, numeroushousekeeping genes and several hypothetical proteins weredetected. In two of the Trichoderma strains (T. asperellumT53 and Trichoderma sp. T78), the most represented gene(T53C94 and T78C171, respectively) was a sequencesimilar to a hydrophobin from H. jecorina. A hydrophobinwas also the most abundantly represented gene in T.harzianum CECT 2413 (Vizcaíno et al. 2006). It was alsopresent as one of the most abundant genes in the libraryLT002 from T. reesei (Diener et al. 2004), but it could notbe found in a similar frequency table of any otherfilamentous fungus. In some species like Magnaporthegrisea (Kim et al. 2001) or Metarhizium anisopliae (StLeger et al. 1992b) hydrophobins have been associated withstress responses like nitrogen or carbon starvation. Recent-ly, it has been described that the hydrophobins I and II fromT. reesei have a role in hyphal development and sporula-tion, respectively (Askolin et al. 2005).

In addition to the hydrophobins, there were other highlyexpressed genes that were found in more than oneTrichoderma strain. First of all, a similar sequence to themajor component of the Woronin bodies (the HEX1protein) was found in T. longibrachiatum T52 (T52C95)and T. virens T59 (T59C215). It was also present as one ofthe most abundant genes in T. harzianum (Liu and Yang2005). It is interesting to note that a similar sequence wasidentified in other Trichoderma species (T. hamatum)during the direct confrontation between Trichodermaand the plant-pathogen fungus Sclerotinia sclerotiorum(Carpenter et al. 2005). In that study, a subtractivehybridization strategy was used to detect genes expressedduring mycoparasitism. In T. reesei, hex1 is highly expressedduring exponential growth (Curach et al. 2004). Additional-ly, in M. grisea, it has been found that hex1 is essential forefficient pathogenesis and for survival under nitrogenstarvation conditions. Curiously, hex1 was induced undernitrogen starvation but limiting the amount of carbon sourcedid not elicit the same effect (Soundararajan et al. 2004).Thus, according to these data, something similar couldhappen in Trichoderma species.

A similar sequence to the chitin-induced fungal cell wallprotein 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 precursorwas detected (Liu and Yang 2005) in T. harzianum as themost represented gene. The role of QID3 is unknown but ithas been proposed that it is a cell wall associated proteinthat is essential for cell–cell attachment. Thus, QID3 maybe functioning in appressorium formation as well aspathogen recognition and attachment (Lora et al. 1995).

Finally, among the shared most expressed genes, a sequencesimilar to the NMT1 protein was identified in T. long-ibrachiatum T52 (T52C94) and T. virens T59 (T59C77). Itwas also identified as a highly expressed gene in T. harzianumCECT 2413 (Vizcaíno et al. 2006), and it is probablyinvolved in the synthesis of thiamine (Morett et al. 2003).

Among the most represented genes found in only one ofthe libraries, first of all, it must be highlighted that acyclophilin (T53C103) was identified in T. asperellum T53.It was not only identified in the frequency tables obtainedin both EST studies carried out in T. harzianum (Liu andYang 2005; Vizcaíno et al. 2006), but also in other fungilike Mycosphaerella graminicola (Keon et al. 2005).Cyclophilins possess peptidyl-prolyl cis–trans isomeraseactivity in vitro and can play roles in a great variety ofprocesses like protein folding and transport, RNA splicing,and the regulation of multiprotein complexes in cells (Wangand Heitman 2005). So far, cyclophilins have not beenextensively studied in filamentous fungi. However, it wasfound that a cyclophilin (CYP1) from M. grisea acts as avirulence determinant in rice blast (Viaud et al. 2002).

Secondly, a sequence similar to the clock controlledprotein 6 (CCG6) from N. crassa (T53C11) was detected inthe same strain. It was present as the second most abundantgene in a study carried out in the wheat pathogen M.graminicola (Keon et al. 2005). In N. crassa, ccg6expression is cyclical, peaking during the late night toearly morning hours. It has been proposed that it is bothdevelopmentally regulated and photoinducible and it couldbe involved in conidiation (Bell-Pedersen et al. 1996).

It is interesting to note that two unique sequences thatcould be involved in the mycoparasitism process were alsodetected as highly abundant genes in T. virens 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 theentomopathogenic fungus M. anisopliae (St Leger et al.1992a). In fact, the efficacy of this fungus as a biologicalcontrol agent can be substantially improved by over-expression of PR1A (St Leger et al. 1996). Curiously, nocell-wall degrading enzyme has been detected in thefrequency tables obtained in both the related to biocontrolcollections 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 antimicrobialactivity. This enzyme is responsible for the first step in thebiosynthesis of the coenzyme A, but also of the pathwayintermediate 4′-phosphopantetheine, an essential prostheticgroup for the activity of a family of enzymes called peptidesynthetases (Kurtov et al. 1999). These large proteins areinvolved in the synthesis of nonribosomal peptides, some ofthem possessing antibiotic activities, and they could also beinvolved in the biocontrol process.

When we performed the annotation of the sequences, theoverall percentage of assigned GO-terms (40.9%) waslower than in T. harzianum CECT 2413 (51.1%) (Vizcaínoet al. 2006), using the same automatic annotation method.A similar percentage of GO-annotated unique sequenceswas only achieved in T. asperellum T53 (51.2%). Thedistribution of the GO terms among the Trichoderma strainswas quite similar, which is logical taking into account thatthe four cDNA libraries were made in similar growthconditions (Table 2).

When we compared the GO-term distribution in theselibraries with the similar study made in the cDNA librariesobtained from T. harzianum CECT 2413 (Vizcaíno et al.2006), the distribution was quite similar to the librariesL02, L03, and L10. However, significant differences werefound between the library L06 and the rest (including thosecoming from this work). The library L06 was made fromTrichoderma growing in solid media and this could be thereason for these changes in the distribution, although otherexplanations cannot be overruled (Vizcaíno et al. 2006). Assome of the growth conditions were shared among theTrichoderma species included in the “TrichoEST” project,and although none of the cDNA libraries made for thepresent work were obtained in identical conditions to thoseused in T. harzianum CECT 2413 (Vizcaíno et al. 2006), itseems logical to find a comparable distribution of the GOterms in all the strains.

Among the strains studied in this work, T. longibrachia-tum T52 is the only one that belongs to the taxonomicalsect. Longibrachiatum, whose members are not consideredto be biocontrol agents (Hermosa et al. 2004). This strain isalso the only one isolated from a nonagricultural sourcebecause it was found in a water treatment plant. In aprevious study (Vizcaíno et al. 2005), we screened theantimicrobial activities in selected isolates representingthree Trichoderma sections. T. longibrachiatum showedthe best nonenzymatic antimicrobial profiles against bacte-ria, yeasts, and filamentous fungi, suggesting that itsantibiotic mechanism could be more relevant than itsglucacolytic/chitinolytic mode of action. Moreover, anisoenzyme study showed that T. longibrachiatum was ableto produce glucanases, chitinases, and proteases. However,

860 Appl Microbiol Biotechnol (2007) 75:853–862

extracellular protein extracts were not effective in theinhibition of the hyphal growth of Botrytis cinerea (Sanzet al. 2004). This could explain our results (both the profileof highly represented genes and the distribution of the GOterms in T. longibrachiatum T52 could be consideredsimilar to the other strains). Because Trichoderma canintegrate several biocontrol mechanisms (Howell 2003), itseems that there are additional environmental factors givingsimilar transcriptomes with different biological functions.The relevance of the strain T. longibrachiatum T52 as abiocontrol agent is now subject to study mainly in base totheir metabolite production with antifungal activity.

The 8,160 ESTs obtained in this study represent themajor attempt so far to define the gene set of theseTrichoderma species. Overall, they represent about 4,480unique sequences from four different Trichoderma strains, afact that dramatically increases the number of identifiedTrichoderma genes, reflecting also the genetic diversitywith this genus.

Acknowledgements First, the authors want to acknowledge thefinancial support of the European Commission to the project“TrichoEST” (QLK3-CT-2002-02032) and the “Fundación RamónAreces”. We want to recognize the work carried out by I. Chamorro,E. Keck, J.A. de Cote, I. González, and M. Andrada for their technicalsupport. Authors want also to acknowledge R. Jiménez, A. Gaignard,M. P. García-Pastor, and J. Heinrich, who helped in differentbioinformatics related tasks. Additionally, we want to thank C.Mungall for his help in setting up the AmiGO browser. Thismanuscript is dedicated to Prof. Antonio Llobell because without hiscontribution, this project could not be carried out.

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