BACKGROUND
trichoderma is a fungal genus that includes cosmopolitan fungi able to colonize different substrates under diverse environmental conditions.
one of the most significant ecological niches occupied by trichoderma species is the plant rhizosphere, which is effectively colonized due to the capacity of these fungi to interact with plants and compete with other soil organisms  <cit> .
this ability is the result of a long period evolution in which biological mechanisms for attacking other microorganisms and for enhancing plant growth have developed in trichoderma  <cit> .
the biocontrol activity of trichoderma depends on its metabolic versatility and secretory potential, which are responsible for the production of large amounts of highly diverse hydrolytic enzymes involved in the degradation of fungal cell walls  <cit> .
since trichoderma species are efficient antagonists of other fungi and due to their ubiquity and rapid substrate colonization, they have been commonly used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry  <cit> .
the trichoderma genome, although it is a fungal genus of high biotechnological value, has been poorly surveyed compared to other microorganisms.
a structural genomics project, carried out by the u.s. joint genome institute  <cit>  has provided the first version of the full genome sequence of the t. reesei strain qm  <dig>  an isolate without known biocontrol abilities, but with industrial interest.
additionally, the functional genomics eu-funded project "trichoest"  <cit>  was undertaken by an international consortium comprised of academic institutions and enterprises.
the aims were to identify genes and gene products from twelve strains with biotechnological value from different trichoderma species  <cit> .
in this project, the antagonistic strain t. harzianum cect  <dig> was selected representing the t. harzianum biotype.
in this work, mrna populations from trichoderma transcribed among others, under mycoparasitic and nutrient stress conditions, trying to simulate some of the environmental conditions that take place in the soil, were cloned as cdnas and were the origin of expressed sequence tags .
this strategy has been used as an efficient and economical approach for large-scale gene discovery, to explore gene regulation patterns and to identify differentially regulated genes.
during the last years, a large number of ests have been generated from several filamentous fungi and oomycetes, including among others gibberella zeae  <cit> , magnaporthe grisea  <cit> , mycosphaerella graminicola  <cit> , phytophthora parasitica  <cit> , uromyces fabae  <cit>  or ustilago maydis  <cit> .
there are several previous studies involving est approaches that have been carried out in trichoderma species.
in the first one  <cit> , the metabolism of t. reesei qm  <dig> was studied focusing on the anaerobic and aerobic degradation of glucose.
their genomic work produced the sequence of  <dig>  randomly selected cdnas corresponding to  <dig>  unique transcripts and the complete mitochondrial genome of t. reesei.
their complete est database and the details on the experimental procedure and results are available on line  <cit> .
in another study  <cit> ,  <dig>  ests from a sole cdna library were sequenced and  <dig>  unique sequences were obtained from t. reesei qm6a.
they also detected twelve enzymes involved in biomass degradation, performed microarray expression analysis and found that these enzymes were transcriptionally regulated  <cit> .
diener et al  <cit>  identified genes from t. reesei qm6a involved in protein processing and secretion.
a total of  <dig>  ests from two different cdna libraries were sequenced, corresponding to  <dig>  unique transcripts.
liu and yang  <cit> , working with a t. harzianum unknown strain, obtained  <dig>  ests  from a single cdna library.
in the most recent study,  <dig>  ests  were retrieved using a subtractive strategy.
two cdna subtraction libraries were used in order to identify genes that were differentially expressed in response to secretion stress in t. reesei rut-c <dig>  <cit> .
in the present study we report the analysis of  <dig>  ests obtained from t. harzianum cect  <dig>  these sequences were derived from eight different cdna libraries that had been made combining different growth conditions.
overall,  <dig>  unique transcripts were identified and go-terms were assigned.
in addition, the relative abundance of ests provided a measure of gene expression.
we identified the putative secreted proteins and also performed a comparative analysis among our dataset and the other collections of ests from trichoderma species.
finally, the t. harzianum ests were compared to genomic sequence databases from several animals, plants and fungi.
RESULTS
est sequence determination and analysis
different conditions were used to build specific and mixed cdna libraries for the "trichoest" project as seen in the methods section.
eight different libraries were made  and est sequences were produced .
a total of  <dig>  sequences were identified as having high quality by phred  <cit>   from the  <dig>  sequencing reactions .
the average sequence length was  <dig> nucleotides, and approximately  <dig> % of the ests were longer than  <dig> nucleotides, while  <dig> % of the sequences were shorter than  <dig> nucleotides.
ests were generated from different libraries with the expectation that more unique genes would be identified from moderate levels of sequencing from diverse libraries rather than deep sequencing of a single library.
the combined ests for all libraries clustered into  <dig> multisequence contigs, with  <dig>  singlets yielding an estimate of  <dig>  unique sequences.
these unique sequences are listed in the additional file  <dig>  the ests that are contained in each contig are listed in the additional file  <dig>
the distribution of ests across multiple libraries was assessed as a measure of uniqueness of gene concurrence across libraries.
no contig contained sequences that were from seven or eight libraries, and only nine unique sequences were present in six libraries.
most of the contigs contained sequences found in one to three libraries, with the largest category being contigs represented by clones from two libraries .
this pattern is mainly due to the fact that many of the contigs contain only a small number of ests.
considering only those contigs representing  <dig> or more ests, it is found that most of the genes are usually represented in multiple libraries.
functional annotation and analysis
unique sequences were assigned functions according to gene ontology  terms  <cit>  based on blast definitions using the program blast2go  <cit> .
go categories were assigned to  <dig>  of the  <dig>  predicted unique sequences .
later, we used a locally implemented amigo browser in order to examine the representation of genes across different functional categories.
our amigo browser is publicly available  <cit> .
however, in this paper we only cite those libraries that provided more than  <dig> unique sequences: l <dig>  l <dig>  l <dig> and l <dig> .
the gene distribution in the main ontology categories 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   <cit> .
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 very similar across the libraries that came from trichoderma grown in liquid media .
however, differences were found in l <dig>  made from trichoderma grown in solid media .
for example, in l <dig> the percentage of go terms in the following categories was significantly higher than in the other libraries: "metabolism" , "biosynthesis" , "cellular metabolism" , "macromolecule metabolism" , "primary metabolism" , "nucleic acid binding" , "structural molecule activity"  and "intracellular" .
additionally, in l <dig> a lower percentage was found in other categories like "catalytic activity" , "hydrolase activity" , "extracellular region"  or "membrane" .
identification of putative secreted proteins
t. harzianum is the source of a number of secreted proteins produced for various industrial applications.
to identify potential secreted proteins we used signalp  <dig>   <cit>  in order to search for predicted proteins with a signal peptide.
we found that  <dig> of these predicted proteins  possessed this putative signal peptide .
exploration of more abundantly expressed genes
sequencing of random cdna clones allows studies of mrna abundance.
thus, analysis of the frequency of specific ests that form individual contigs can provide information with respect to the expression levels of particular genes under different experimental conditions  <cit> .
table  <dig> displays the total number of contigs made up of  <dig> or more ests together with the originating libraries.
the data illustrate the functional diversity of these highly expressed unique sequences with apparently no particular functional category dominating the analysis.
however, at this point it must be considered that a significant amount of hypothetical and/or unassigned-function proteins were also detected.
as expected, a number of housekeeping genes involved in protein translation, carbon metabolism and energy production were identified, as some of the hits corresponded to genes like the translation elongation factor 1α, histones h <dig> and h <dig>  polyubiquitin, glyceraldehyde 3-phosphate dehydrogenase or ribosomal proteins.
the most abundantly represented gene in the total collection  was similar to a hydrophobin from hypocrea jecorina .
hydrophobins are small molecular weight proteins of fungal origin that can play roles in diverse physiological processes including adhesion, development and pathogenesis  <cit> .
we also found genes probably involved in the synthesis of thiamine : t34c <dig>  and t34c <dig>  were similar to the nmt <dig> protein and t34c <dig>  was similar to the thi <dig> protein, a thiazole biosynthetic enzyme.
additionally, we also detected sequences similar to an adp/atp carrier protein , the atp synthase protein  <dig>   <cit> , a peptide involved in stress response  and a cyclophilin .
comparison to the nr database and interproscan annotation
sequence comparison using the blastx algorithm against the ncbi non redundant  database allowed the identification of  <dig>  unique sequences .
thus,  <dig> sequences did not exhibit significant similarity  to genes in the nr database.
additional information on the ests was obtained by protein-signature scanning.
interproscan was used for sequence comparison to the interpro database  <cit> .
this database contains signature information from hidden markov models, regular expressions, fingerprints and profiles for protein families, and domains from public domain database projects including pfam  <cit> , prosite  <cit> , prodom  <cit> , prints  <cit> , tigrfams  <cit>  and smart  <cit> .
the submission facilitated the annotation of  <dig>   unique sequences with significant protein signatures.
within them,  <dig>   sequences contained associated interpro  numbers.
of these  <dig>  sequences,  <dig> had not been annotated during the sequence comparison against the ncbi nr database.
these data are included in the additional file  <dig>
comparison with other trichoderma collections of ests
we used the tblastx algorithm  <cit>  in order to study the presence of similar sequences in the collections of ests from trichoderma publicly available.
at the e-value < 10- <dig> level, the highest percentages of similar sequences were found in three collections from t. reesei:  <dig> %  <cit> ,  <dig> %  <cit>  and  <dig> %  <cit> .
unexpectedly, only  <dig> % of similar sequences was found in the related to biocontrol collection obtained from t. harzianum  <cit> .
finally, a very low percentage  of similar sequences was found with the ests recently obtained from t. reesei following a subtractive strategy  <cit> .
blast analysis to species sequence datasets
we used the blastx algorithm  <cit>  to identify ests with sequence similarity to the protein sequences derived from the genomic sequence datasets of  <dig> eukaryotic species .
we used an e-value < 10- <dig> as indicative of sequence similarity significance.
in these comparisons it is important to note that the t. harzianum sequences do not represent the complete genome.
all unique sequences from t. harzianum were queried against the sequence datasets.
a table listing the number of unique sequences possessing a top hit below the e-values of 10- <dig>  10- <dig>  10- <dig>  10- <dig>  and 10- <dig> was produced .
of the  <dig>  unique sequences from t. harzianum, the highest proportion of similar sequences was found in filamentous fungi.
at the 10- <dig> level, the percentages were: t. reesei , fusarium graminearum , neurospora crassa , m. grisea  and aspergillus nidulans .
these proportions decreased in the basidiomicete u. maydis  and cryptococcus neoformans  and also in the yeasts candida albicans , schizosaccharomyces pombe  and saccharomyces cerevisiae .
at this level, a total percentage of an  <dig> % of the unique sequences had sequence similarity with at least one of the fungal datasets.
the number of ests plus contigs with similarity to sequences of plants was slightly higher  than what was found in animals .
a total of  <dig>  unique sequences  showed sequence similarity with at least one species with an e-value < 10- <dig>  logically, these percentages decreased as the e-value increased .
for instance, at the e < 10- <dig> level,  <dig> % of the sequences had similar sequences in t. reesei,  <dig> % in f. graminearum and  <dig> % in a. nidulans, being the rest of the genomes not significantly represented.
further analysis of the t. harzianum unique sequences grouped these sequences based upon the eukaryotic taxa in which sequence similarity was found.
the classification of unique sequences conserved across all eukaryotes was defined by at least one similar sequence in each of the fungi, plants and animals.
under these criteria,  <dig> of the  <dig>  unique sequences  that showed sequence similarity  with at least one organism, were present in all eukaryotic taxa .
these sequences are listed in the additional file  <dig>
moreover,  <dig>  unique sequences were found in genomes from fungi and animals  and a slightly higher number of genes  was shared between fungi and plants .
furthermore, the number of unique sequences with similarity to the fungal and plant genomes but not the animal genomes was  <dig>  meanwhile, the number of unique sequences with similar sequences in the fungal plus in either of the two animal, but not in the plant genomes was  <dig> .
the t. harzianum unique sequences found to have similar sequences only in fungal species totalled  <dig>  .
these sequences are also listed in the additional file  <dig>
the number of orphan unique sequences  was  <dig> .
however, the number of unique sequences lacking a similarity to sequences in the nr database was  <dig> .
thus, after our cross-species identification of similar sequences, we reduced the number of orphan genes by another  <dig> unique sequences.
discussion
we are investigating the genome of t. harzianum cect  <dig>  a strain with a great interest in biotechnology and biological control  <cit> .
est sequencing provides information about functional identification of genes, gene structure and gene expression patterns.
like in other recent studies carried out in fungi  <cit> , a strategy of sequencing a variety of different libraries was used to maximize the number of unique genes.
the approach based on the construction of cdna libraries from a mixture of conditions was successfully used in t. reesei by foreman et al.
<cit> .
the trichoderma growth conditions for the different cdna libraries were mainly chosen in order to simulate in vitro some aspects of the biocontrol process occurring in the soil environment like mycoparasitism  <cit> .
this strategy has proved to be successful in order to identify genes involved in the biocontrol such as thptr <dig> , a peptide transporter recently studied by our group that was highly expressed when trichoderma was interacting with the plant-pathogenic fungus botrytis cinerea  <cit> .
additionally, similar findings have been obtained for other genes like erg <dig>  involved in the biosynthesis of terpenes  <cit>  or the proteases pra <dig>  <cit>  and p <dig>  <cit> .
overall, we searched in our 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 , glucanase  or protease  activities.
this isoenzyme multiplicity has been described before by different groups and reported by our team in a study where several isoenzymes with glucanase, protease or chitinase activities were detected in different trichoderma species, including t. harzianum cect  <dig>  <cit> .
sequence redundancy within our study was  <dig> %.
this level of redundancy was lower than in the two comparable studies carried out in t. reesei:  <dig> %  <cit>  and  <dig> %  <cit> .
in the first one,  <dig>  ests were sequenced from a single cdna library made from a mixture of more than  <dig> different growth conditions.
however, in the second one  <cit> , a unique growth condition was used and the number of sequenced ests was also lower .
the subtractive study  <cit>  had a sequence redundancy of  <dig> %.
finally, the remaining study is not comparable since a separate assembly process was used for each library  <cit> .
taking into account that sequence redundancy increases with the total number of sequenced ests, we consider that our strategy, designed in order to maximize the number of unique genes without increasing in excess the number of sequences, worked properly.
overall,  <dig>  unique sequences were identified in t. harzianum cect  <dig>  which represents a significant portion of the genome.
according to recent available data  <cit> , the number of genes in the t. reesei genome  is very close to  <dig> .
this is similar to what has been found in other filamentous fungi.
thus, our collection of genes would represent about one third of the total.
blastx searches indicated that  <dig> % of the unique sequences had sequence similarity to an entry in the ncbi nr database.
this percentage is quite similar to what has been recently found in a similar study in aspergillus niger   <cit> , but it is much higher than what was found in other recent est studies carried out in other fungi like conidiobolus coronatus   <cit> , phakospora pachyrhizi   <cit>  and u. maydis   <cit> .
new sequence data from complete genome projects from several fungi had made possible this increase although a high proportion of the hits remains annotated as hypothetical proteins.
the degree of annotation was extended through the identification of protein motifs, using interproscan searches of the interpro database.
this extension resulted in annotation of a  <dig> %  of the unique sequences, including  <dig>  with interpro annotation.
this percentage is again much higher than the one found in u. maydis   <cit> .
as seen before, in a number of cases clones that did not cluster  displayed sequence similarity to the same protein.
several factors can account for this.
among them,  the clones are different genes that are homologs of the same protein,  the clones align with different regions of the same search hit but do not overlap  with each other, and  the clones represent different splice variants of the same gene.
we searched for alternative transcript forms by looking carefully inside each contig but apparently, none of them were detected in the present study.
they have been recently found in other filamentous fungi like m. grisea  <cit>  and fusarium verticillioides  <cit> , although perhaps the most extensive genome-wide survey on alternative splicing in a fungus comes from the basidiomycete c. neoformans serotype d  <cit> , where this mechanism was found in  <dig> % of the genes.
additionally, there are at least two studies where alternative splicing has been described in trichoderma species.
in the first one, carried out also in t. harzianum cect  <dig>  two alternative forms of a glucoamylase gene were detected in different growth conditions  <cit> .
in the second one, the authors found two different mrna species from two chitinases , depending also on the culture media where t. atroviride had been previously grown  <cit> .
est abundance
the analysis of the relative abundance of individual ests that make up unique sequences  from different libraries can be used as a first indicator of transcript abundance.
we identified a number of unique sequences generated from  <dig> or more ests .
apparently, no particular gene family was predominant.
this agrees with some previous works  <cit> , but it is in contrast with other analysis where particular classes of genes  dominated these frequency tables  <cit> .
however, numerous housekeeping genes were detected as expected.
a unique sequence similar to the hydrophobin ii from h. jecorina was the most represented gene.
a similar sequence  was also found as one of the most expressed genes in the library lt <dig> from t. reesei  <cit> .
however, we have not found another possible hydrophobin in a similar frequency table made in any other fungus including t. harzianum  <cit> .
in a recent study  <cit> , it was found that the t. reesei hydrophobins i and ii had a role in hyphal development and sporulation, respectively.
among the most highly expressed genes we also detected sequences that could be involved in thiamine biosynthesis, similar to the nmt <dig> and thi <dig> proteins respectively.
thi <dig> is a thiazole biosynthetic enzyme that had been previously found  as highly expressed in t. harzianum  <cit> .
however, both genes could also be involved in other processes  <cit> .
in s. cerevisiae thi <dig> appears to be a dual function protein involved in thiazole biosynthesis and tolerance to mitochondrial dna damage  <cit> .
a putative cyclophilin was also identified as one of the most abundant transcripts.
cyclophilins include the binding proteins of the cyclic peptide cyclosporin a, they possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, rna splicing and the regulation of multi-protein complexes in cells  <cit> .
one similar sequence was also found as one of the most expressed genes  in the est collection from t. harzianum  <cit> .
so far, cyclophilins have been more studied in yeasts  <cit>  than in filamentous fungi  <cit> .
go terms
the percentage of assigned go-terms  was slightly higher than in one similar study in a. niger  <cit> .
as far as we know, this is the first time that the program blast2go  <cit>  has been used for this purpose.
clear differences were found in the distribution of the go terms among the gene library l <dig> and the other three .
this could be explained considering that l <dig> was the only library among them made using mrna from trichoderma growing in solid medium.
however, it must be also considered that these differences could arise more from their different composition than from the solid or liquid nature of the media.
the liquid media included different stress-related growth conditions like nitrogen or carbon starvation, chitin or fungal cell walls as sole carbon source whereas the solid medium covered only carbon starvation.
we identified  <dig> predicted proteins with a putative signal peptide.
gene ontology annotation categorized only  <dig> of the predicted proteins as "extracellular", included in the go terms "extracellular matrix"  and "extracellular region"  .
however, it must be taken into account that we were able to assign a go term to  <dig> % of the unique sequences.
the percentage of predicted proteins with signal peptide  was almost identical to what was found in a. niger   <cit>  but lower than in t. reesei , using the same algorithm  <cit> .
these differences could be related to the different growth conditions from which the cdna libraries were made or to the fact that they are different species.
comparison with other trichoderma collections of ests
we found that only  <dig> % of the unique sequences had similar sequences in the related to biocontrol collection obtained from t. harzianum  <cit> .
a higher number of similar sequences was found in the other comparable collections from t. reesei, specifically in the collection of ests involved in protein processing and secretion  <cit> .
these results were unexpected because in principle, a highest number of similar sequences should have been found in the est collection related to biocontrol  <cit>  than in the t. reesei collections.
the lack of information on the strain and the growth conditions in which that est collection for t. harzianum was obtained  <cit>  makes difficult to explain this fact.
as for the differences in the presence of similar sequences between the three comparable collections from t. reesei, it is logical that the lowest percentage of similar sequences was found in a collection obtained in very different growth conditions, with glycerol as sole carbon source  <cit> , than the ones used in our study.
blast analysis to species sequence datasets
the unique sequences were compared to the genomes of eleven different fungi, two animals  and two plants .
within the total of  <dig>  unique sequences  that showed sequence similarity with at least one species ,  <dig>   had a similar sequence in the t. reesei complete genome.
it must be considered that many gaps are still present in the current publicly available version  of this genome  <cit> .
the new version  containing very few gaps will be available soon  <cit>  and will allow us to further study the unique sequences that could be found in t. harzianum but not present in t. reesei.
behind t. reesei, the ascomycete f. graminearum possessed the largest number of similar sequences to the t. harzianum unique sequences.
this is not surprising due to the close taxonomic relationship between the trichoderma and fusarium genera, because their teleomorphs are located in the close families nectriaceae and hypocreaceae, respectively  <cit> .
in the species-by-species comparison,  <dig>  unique sequences were present in all the eukaryotic taxa.
a similar percentage  was found in a similar study in u. maydis  <cit> .
these  <dig> clones constitute a  <dig> % of the unique sequences  that showed sequence similarity  with at least one organism .
this number is perhaps slightly low because it has been described that about 40% of the total genes in an eukaryotic genome may be shared with other eukaryotes of different kingdoms, although this does not mean that they are all essential genes  <cit> .
this slightly low percentage could be due to the fact that our est data set constitutes only a portion of the genome.
t. harzianum sequences found to have similar sequences only in fungal species totalled  <dig>  .
this percentage is much higher than what was found in u. maydis   <cit> .
there may be genes among these that have retained phylogenetic signatures dating to the separation of fungi and animals, or genes with signatures representing further changes leading to the current state of t. harzianum.
CONCLUSIONS
the  <dig>  ests identified in this study represent the major attempt so far to define the t. harzianum gene set and represent about  <dig>  genes.
thus, these data dramatically increase the number of identified t. harzianum genes.
the clone collection offers a base for expression profiling, enabling the identification of genes involved in specific physiological processes.
the application of these results is not only of a great interest in biocontrol of plant-pathogens but also in the searching of genes with high biotechnological value.

 trichoderma is a fungal genus that includes cosmopolitan fungi able to colonize different substrates under diverse environmental conditions. one of the most significant ecological niches occupied by trichoderma species is the plant rhizosphere, which is effectively colonized due to the capacity of these fungi to interact with plants and compete with other soil organisms. this ability is the result of a long period evolution in which biological mechanisms for attacking other microorganisms and for enhancing plant growth have developed in trichoderma. the biocontrol activity of trichoderma depends on its metabolic versatility and secretory potential, which are responsible for the production of large amounts of highly diverse hydrolytic enzymes involved in the degradation of fungal cell walls. since trichoderma species are efficient antagonists of other fungi and due to their ubiquity and rapid substrate colonization, they have been commonly used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. the trichoderma genome, although it is a fungal genus of high biotechnological value, has been poorly surveyed compared to other microorganisms. a structural genomics project, carried out by the u.s. joint genome institute has provided the first version of the full genome sequence of the t. reesei strain qm an isolate without known biocontrol abilities, but with industrial interest. additionally, the functional genomics eu-funded project "trichoest" was undertaken by an international consortium comprised of academic institutions and enterprises. the aims were to identify genes and gene products from twelve strains with biotechnological value from different trichoderma species. in this project, the antagonistic strain t. harzianum cect was selected representing the t. harzianum biotype. in this work, mrna populations from trichoderma transcribed among others, under mycoparasitic and nutrient stress conditions, trying to simulate some of the environmental conditions that take place in the soil, were cloned as cdnas and were the origin of expressed sequence tags. this strategy has been used as an efficient and economical approach for large-scale gene discovery, to explore gene regulation patterns and to identify differentially regulated genes. during the last years, a large number of ests have been generated from several filamentous fungi and oomycetes, including among others gibberella zeae, magnaporthe grisea, mycosphaerella graminicola, phytophthora parasitica, uromyces fabae or ustilago maydis. there are several previous studies involving est approaches that have been carried out in trichoderma species. in the first one, the metabolism of t. reesei qm was studied focusing on the anaerobic and aerobic degradation of glucose. their genomic work produced the sequence of randomly selected cdnas corresponding to unique transcripts and the complete mitochondrial genome of t. reesei. their complete est database and the details on the experimental procedure and results are available on line. in another study, ests from a sole cdna library were sequenced and unique sequences were obtained from t. reesei qm6a. they also detected twelve enzymes involved in biomass degradation, performed microarray expression analysis and found that these enzymes were transcriptionally regulated. diener et al identified genes from t. reesei qm6a involved in protein processing and secretion. a total of ests from two different cdna libraries were sequenced, corresponding to unique transcripts. liu and yang, working with a t. harzianum unknown strain, obtained ests from a single cdna library. in the most recent study, ests were retrieved using a subtractive strategy. two cdna subtraction libraries were used in order to identify genes that were differentially expressed in response to secretion stress in t. reesei rut-c. in the present study we report the analysis of ests obtained from t. harzianum cect these sequences were derived from eight different cdna libraries that had been made combining different growth conditions. overall, unique transcripts were identified and go-terms were assigned. in addition, the relative abundance of ests provided a measure of gene expression. we identified the putative secreted proteins and also performed a comparative analysis among our dataset and the other collections of ests from trichoderma species. finally, the t. harzianum ests were compared to genomic sequence databases from several animals, plants and fungi.  est sequence determination and analysis different conditions were used to build specific and mixed cdna libraries for the "trichoest" project as seen in the methods section. eight different libraries were made and est sequences were produced. a total of sequences were identified as having high quality by phred from the sequencing reactions. the average sequence length was nucleotides, and approximately % of the ests were longer than nucleotides, while % of the sequences were shorter than nucleotides. ests were generated from different libraries with the expectation that more unique genes would be identified from moderate levels of sequencing from diverse libraries rather than deep sequencing of a single library. the combined ests for all libraries clustered into multisequence contigs, with singlets yielding an estimate of unique sequences. these unique sequences are listed in the additional file the ests that are contained in each contig are listed in the additional file the distribution of ests across multiple libraries was assessed as a measure of uniqueness of gene concurrence across libraries. no contig contained sequences that were from seven or eight libraries, and only nine unique sequences were present in six libraries. most of the contigs contained sequences found in one to three libraries, with the largest category being contigs represented by clones from two libraries. this pattern is mainly due to the fact that many of the contigs contain only a small number of ests. considering only those contigs representing or more ests, it is found that most of the genes are usually represented in multiple libraries. functional annotation and analysis unique sequences were assigned functions according to gene ontology terms based on blast definitions using the program blast2go. go categories were assigned to of the predicted unique sequences. later, we used a locally implemented amigo browser in order to examine the representation of genes across different functional categories. our amigo browser is publicly available. however, in this paper we only cite those libraries that provided more than unique sequences: l l l and l. the gene distribution in the main ontology categories 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. 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 very similar across the libraries that came from trichoderma grown in liquid media. however, differences were found in l made from trichoderma grown in solid media. for example, in l the percentage of go terms in the following categories was significantly higher than in the other libraries: "metabolism", "biosynthesis", "cellular metabolism", "macromolecule metabolism", "primary metabolism", "nucleic acid binding", "structural molecule activity" and "intracellular". additionally, in l a lower percentage was found in other categories like "catalytic activity", "hydrolase activity", "extracellular region" or "membrane". identification of putative secreted proteins t. harzianum is the source of a number of secreted proteins produced for various industrial applications. to identify potential secreted proteins we used signalp in order to search for predicted proteins with a signal peptide. we found that of these predicted proteins possessed this putative signal peptide. exploration of more abundantly expressed genes sequencing of random cdna clones allows studies of mrna abundance. thus, analysis of the frequency of specific ests that form individual contigs can provide information with respect to the expression levels of particular genes under different experimental conditions. table displays the total number of contigs made up of or more ests together with the originating libraries. the data illustrate the functional diversity of these highly expressed unique sequences with apparently no particular functional category dominating the analysis. however, at this point it must be considered that a significant amount of hypothetical and/or unassigned-function proteins were also detected. as expected, a number of housekeeping genes involved in protein translation, carbon metabolism and energy production were identified, as some of the hits corresponded to genes like the translation elongation factor 1α, histones h and h polyubiquitin, glyceraldehyde 3-phosphate dehydrogenase or ribosomal proteins. the most abundantly represented gene in the total collection was similar to a hydrophobin from hypocrea jecorina. hydrophobins are small molecular weight proteins of fungal origin that can play roles in diverse physiological processes including adhesion, development and pathogenesis. we also found genes probably involved in the synthesis of thiamine: t34c and t34c were similar to the nmt protein and t34c was similar to the thi protein, a thiazole biosynthetic enzyme. additionally, we also detected sequences similar to an adp/atp carrier protein, the atp synthase protein, a peptide involved in stress response and a cyclophilin. comparison to the nr database and interproscan annotation sequence comparison using the blastx algorithm against the ncbi non redundant database allowed the identification of unique sequences. thus, sequences did not exhibit significant similarity to genes in the nr database. additional information on the ests was obtained by protein-signature scanning. interproscan was used for sequence comparison to the interpro database. this database contains signature information from hidden markov models, regular expressions, fingerprints and profiles for protein families, and domains from public domain database projects including pfam, prosite, prodom, prints, tigrfams and smart. the submission facilitated the annotation of unique sequences with significant protein signatures. within them, sequences contained associated interpro numbers. of these sequences, had not been annotated during the sequence comparison against the ncbi nr database. these data are included in the additional file comparison with other trichoderma collections of ests we used the tblastx algorithm in order to study the presence of similar sequences in the collections of ests from trichoderma publicly available. at the e-value < 10- level, the highest percentages of similar sequences were found in three collections from t. reesei: %, % and %. unexpectedly, only % of similar sequences was found in the related to biocontrol collection obtained from t. harzianum. finally, a very low percentage of similar sequences was found with the ests recently obtained from t. reesei following a subtractive strategy. blast analysis to species sequence datasets we used the blastx algorithm to identify ests with sequence similarity to the protein sequences derived from the genomic sequence datasets of eukaryotic species. we used an e-value < 10- as indicative of sequence similarity significance. in these comparisons it is important to note that the t. harzianum sequences do not represent the complete genome. all unique sequences from t. harzianum were queried against the sequence datasets. a table listing the number of unique sequences possessing a top hit below the e-values of 10- 10- 10- 10- and 10- was produced. of the unique sequences from t. harzianum, the highest proportion of similar sequences was found in filamentous fungi. at the 10- level, the percentages were: t. reesei, fusarium graminearum, neurospora crassa, m. grisea and aspergillus nidulans. these proportions decreased in the basidiomicete u. maydis and cryptococcus neoformans and also in the yeasts candida albicans, schizosaccharomyces pombe and saccharomyces cerevisiae. at this level, a total percentage of an % of the unique sequences had sequence similarity with at least one of the fungal datasets. the number of ests plus contigs with similarity to sequences of plants was slightly higher than what was found in animals. a total of unique sequences showed sequence similarity with at least one species with an e-value < 10- logically, these percentages decreased as the e-value increased. for instance, at the e < 10- level, % of the sequences had similar sequences in t. reesei, % in f. graminearum and % in a. nidulans, being the rest of the genomes not significantly represented. further analysis of the t. harzianum unique sequences grouped these sequences based upon the eukaryotic taxa in which sequence similarity was found. the classification of unique sequences conserved across all eukaryotes was defined by at least one similar sequence in each of the fungi, plants and animals. under these criteria, of the unique sequences that showed sequence similarity with at least one organism, were present in all eukaryotic taxa. these sequences are listed in the additional file moreover, unique sequences were found in genomes from fungi and animals and a slightly higher number of genes was shared between fungi and plants. furthermore, the number of unique sequences with similarity to the fungal and plant genomes but not the animal genomes was meanwhile, the number of unique sequences with similar sequences in the fungal plus in either of the two animal, but not in the plant genomes was. the t. harzianum unique sequences found to have similar sequences only in fungal species totalled. these sequences are also listed in the additional file the number of orphan unique sequences was. however, the number of unique sequences lacking a similarity to sequences in the nr database was. thus, after our cross-species identification of similar sequences, we reduced the number of orphan genes by another unique sequences. discussion we are investigating the genome of t. harzianum cect a strain with a great interest in biotechnology and biological control. est sequencing provides information about functional identification of genes, gene structure and gene expression patterns. like in other recent studies carried out in fungi, a strategy of sequencing a variety of different libraries was used to maximize the number of unique genes. the approach based on the construction of cdna libraries from a mixture of conditions was successfully used in t. reesei by foreman et al.. the trichoderma growth conditions for the different cdna libraries were mainly chosen in order to simulate in vitro some aspects of the biocontrol process occurring in the soil environment like mycoparasitism. this strategy has proved to be successful in order to identify genes involved in the biocontrol such as thptr, a peptide transporter recently studied by our group that was highly expressed when trichoderma was interacting with the plant-pathogenic fungus botrytis cinerea. additionally, similar findings have been obtained for other genes like erg involved in the biosynthesis of terpenes or the proteases pra and p. overall, we searched in our 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, glucanase or protease activities. this isoenzyme multiplicity has been described before by different groups and reported by our team in a study where several isoenzymes with glucanase, protease or chitinase activities were detected in different trichoderma species, including t. harzianum cect. sequence redundancy within our study was %. this level of redundancy was lower than in the two comparable studies carried out in t. reesei: % and %. in the first one, ests were sequenced from a single cdna library made from a mixture of more than different growth conditions. however, in the second one, a unique growth condition was used and the number of sequenced ests was also lower. the subtractive study had a sequence redundancy of %. finally, the remaining study is not comparable since a separate assembly process was used for each library. taking into account that sequence redundancy increases with the total number of sequenced ests, we consider that our strategy, designed in order to maximize the number of unique genes without increasing in excess the number of sequences, worked properly. overall, unique sequences were identified in t. harzianum cect which represents a significant portion of the genome. according to recent available data, the number of genes in the t. reesei genome is very close to. this is similar to what has been found in other filamentous fungi. thus, our collection of genes would represent about one third of the total. blastx searches indicated that % of the unique sequences had sequence similarity to an entry in the ncbi nr database. this percentage is quite similar to what has been recently found in a similar study in aspergillus niger, but it is much higher than what was found in other recent est studies carried out in other fungi like conidiobolus coronatus, phakospora pachyrhizi and u. maydis. new sequence data from complete genome projects from several fungi had made possible this increase although a high proportion of the hits remains annotated as hypothetical proteins. the degree of annotation was extended through the identification of protein motifs, using interproscan searches of the interpro database. this extension resulted in annotation of a % of the unique sequences, including with interpro annotation. this percentage is again much higher than the one found in u. maydis. as seen before, in a number of cases clones that did not cluster displayed sequence similarity to the same protein. several factors can account for this. among them, the clones are different genes that are homologs of the same protein, the clones align with different regions of the same search hit but do not overlap with each other, and the clones represent different splice variants of the same gene. we searched for alternative transcript forms by looking carefully inside each contig but apparently, none of them were detected in the present study. they have been recently found in other filamentous fungi like m. grisea and fusarium verticillioides, although perhaps the most extensive genome-wide survey on alternative splicing in a fungus comes from the basidiomycete c. neoformans serotype d, where this mechanism was found in % of the genes. additionally, there are at least two studies where alternative splicing has been described in trichoderma species. in the first one, carried out also in t. harzianum cect two alternative forms of a glucoamylase gene were detected in different growth conditions. in the second one, the authors found two different mrna species from two chitinases, depending also on the culture media where t. atroviride had been previously grown. est abundance the analysis of the relative abundance of individual ests that make up unique sequences from different libraries can be used as a first indicator of transcript abundance. we identified a number of unique sequences generated from or more ests. apparently, no particular gene family was predominant. this agrees with some previous works, but it is in contrast with other analysis where particular classes of genes dominated these frequency tables. however, numerous housekeeping genes were detected as expected. a unique sequence similar to the hydrophobin ii from h. jecorina was the most represented gene. a similar sequence was also found as one of the most expressed genes in the library lt from t. reesei. however, we have not found another possible hydrophobin in a similar frequency table made in any other fungus including t. harzianum. in a recent study, it was found that the t. reesei hydrophobins i and ii had a role in hyphal development and sporulation, respectively. among the most highly expressed genes we also detected sequences that could be involved in thiamine biosynthesis, similar to the nmt and thi proteins respectively. thi is a thiazole biosynthetic enzyme that had been previously found as highly expressed in t. harzianum. however, both genes could also be involved in other processes. in s. cerevisiae thi appears to be a dual function protein involved in thiazole biosynthesis and tolerance to mitochondrial dna damage. a putative cyclophilin was also identified as one of the most abundant transcripts. cyclophilins include the binding proteins of the cyclic peptide cyclosporin a, they possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, rna splicing and the regulation of multi-protein complexes in cells. one similar sequence was also found as one of the most expressed genes in the est collection from t. harzianum. so far, cyclophilins have been more studied in yeasts than in filamentous fungi. go terms the percentage of assigned go-terms was slightly higher than in one similar study in a. niger. as far as we know, this is the first time that the program blast2go has been used for this purpose. clear differences were found in the distribution of the go terms among the gene library l and the other three. this could be explained considering that l was the only library among them made using mrna from trichoderma growing in solid medium. however, it must be also considered that these differences could arise more from their different composition than from the solid or liquid nature of the media. the liquid media included different stress-related growth conditions like nitrogen or carbon starvation, chitin or fungal cell walls as sole carbon source whereas the solid medium covered only carbon starvation. we identified predicted proteins with a putative signal peptide. gene ontology annotation categorized only of the predicted proteins as "extracellular", included in the go terms "extracellular matrix" and "extracellular region". however, it must be taken into account that we were able to assign a go term to % of the unique sequences. the percentage of predicted proteins with signal peptide was almost identical to what was found in a. niger but lower than in t. reesei, using the same algorithm. these differences could be related to the different growth conditions from which the cdna libraries were made or to the fact that they are different species. comparison with other trichoderma collections of ests we found that only % of the unique sequences had similar sequences in the related to biocontrol collection obtained from t. harzianum. a higher number of similar sequences was found in the other comparable collections from t. reesei, specifically in the collection of ests involved in protein processing and secretion. these results were unexpected because in principle, a highest number of similar sequences should have been found in the est collection related to biocontrol than in the t. reesei collections. the lack of information on the strain and the growth conditions in which that est collection for t. harzianum was obtained makes difficult to explain this fact. as for the differences in the presence of similar sequences between the three comparable collections from t. reesei, it is logical that the lowest percentage of similar sequences was found in a collection obtained in very different growth conditions, with glycerol as sole carbon source, than the ones used in our study. blast analysis to species sequence datasets the unique sequences were compared to the genomes of eleven different fungi, two animals and two plants. within the total of unique sequences that showed sequence similarity with at least one species, had a similar sequence in the t. reesei complete genome. it must be considered that many gaps are still present in the current publicly available version of this genome. the new version containing very few gaps will be available soon and will allow us to further study the unique sequences that could be found in t. harzianum but not present in t. reesei. behind t. reesei, the ascomycete f. graminearum possessed the largest number of similar sequences to the t. harzianum unique sequences. this is not surprising due to the close taxonomic relationship between the trichoderma and fusarium genera, because their teleomorphs are located in the close families nectriaceae and hypocreaceae, respectively. in the species-by-species comparison, unique sequences were present in all the eukaryotic taxa. a similar percentage was found in a similar study in u. maydis. these clones constitute a % of the unique sequences that showed sequence similarity with at least one organism. this number is perhaps slightly low because it has been described that about 40% of the total genes in an eukaryotic genome may be shared with other eukaryotes of different kingdoms, although this does not mean that they are all essential genes. this slightly low percentage could be due to the fact that our est data set constitutes only a portion of the genome. t. harzianum sequences found to have similar sequences only in fungal species totalled. this percentage is much higher than what was found in u. maydis. there may be genes among these that have retained phylogenetic signatures dating to the separation of fungi and animals, or genes with signatures representing further changes leading to the current state of t. harzianum.  the ests identified in this study represent the major attempt so far to define the t. harzianum gene set and represent about genes. thus, these data dramatically increase the number of identified t. harzianum genes. the clone collection offers a base for expression profiling, enabling the identification of genes involved in specific physiological processes. the application of these results is not only of a great interest in biocontrol of plant-pathogens but also in the searching of genes with high biotechnological value. 
different growth conditions involving mainly different nutrient conditions and/or stresses were used. eight different cdna libraries from t. harzianum strain cect were constructed. this est collection and its annotation provide a significant resource for basic and applied research on t. harzianum, a fungus with a high biotechnological interest. later, based on the est abundance, we examined the highly expressed genes and a hydrophobin was identified as the gene expressed at the highest level. we compared our collection of ests with the previous collections obtained from trichoderma species and we also compared our sequence set with different complete eukaryotic genomes from several animals, plants and fungi. accordingly, the presence of similar sequences in different kingdoms was also studied. in order to study the genome of this fungus, a functional genomics project called "trichoest" was developed to give insights into genes involved in biological control activities using an approach based on the generation of expressed sequence tags. a total of unique sequences were identified of which % had sequence similarity with genbank entries, using the blastx algorithm. using the gene ontology hierarchy, we performed the annotation of % of the unique sequences and compared its distribution among the gene libraries.
 the filamentous fungus trichoderma harzianum is used as biological control agent of several plant-pathogenic fungi. in order to study the genome of this fungus, a functional genomics project called "trichoest" was developed to give insights into genes involved in biological control activities using an approach based on the generation of expressed sequence tags.  eight different cdna libraries from t. harzianum strain cect were constructed. different growth conditions involving mainly different nutrient conditions and/or stresses were used. we here present the analysis of the ests generated. a total of unique sequences were identified of which % had sequence similarity with genbank entries, using the blastx algorithm. using the gene ontology hierarchy, we performed the annotation of % of the unique sequences and compared its distribution among the gene libraries. additionally, the interproscan algorithm was used in order to further characterize the sequences. the identification of the putatively secreted proteins was also carried out. later, based on the est abundance, we examined the highly expressed genes and a hydrophobin was identified as the gene expressed at the highest level. we compared our collection of ests with the previous collections obtained from trichoderma species and we also compared our sequence set with different complete eukaryotic genomes from several animals, plants and fungi. accordingly, the presence of similar sequences in different kingdoms was also studied.  this est collection and its annotation provide a significant resource for basic and applied research on t. harzianum, a fungus with a high biotechnological interest. 