It has been demonstrated that the cell wall of the fungal pathogen can simulate some aspects of the mycoparasitic interactions between biocontrol fungi and its targets [10
Only a limited amount of overlap (333 unigenes) was observed in both libraries. A total of 1138 and 691 unigenes were unique and only expressed in C1 or C2, respectively. The lack of significant overlap between the individual libraries also suggests a high level of flexibility at the level of gene expression under the examined conditions, some of which may reflect particular requirements for phases of mycoparasitism.
The analysis of the frequency of specific ESTs that form individual contigs can give information about the expression levels of particular genes under different experimental conditions [11
]. The most abundant transcripts in library C2 but no expression in C1 were MFS monosaccharide transporters. MFS transporters transport uni-, sym-, and antiporters of sugars, peptides, drugs, and organic and inorganic ions with 12 or 14 transmembrane spanners [12
]. In the present study, the high proportion of ESTs expressing a homology to MFS monosaccharide transporters implies that they may be responsible for transport of monosaccharides derived from the degradation of RsCW. This was not consistent with the results of a study of T. harzianum
CECT 2413 [13
], in which abundant expression of peptide transporter 2 (PTR2) was found in a cDNA library of T. harzianum
CECT 2413 when interacted directly with Botrytis cinerea
. However, only one EST similar to PTR2 (DV547977
) was detected in library C2. We speculate that this may have been caused by the different cultivation times of the two fungi.
Comparison analysis illustrated variations in the proportions of different pathways. Metabolic pathways of ubiquinone biosynthesis; electron transport and oxidative phosphorylation; purine metabolism; pyrimidine metabolism; alanine and aspartate metabolism; valine, leucine, and isoleucine biosynthesis; porphyrin and chlorophyll metabolism were proportionately more represented in library C1. In contrast, pentose and glucuronate interconversions; fructose and mannose metabolism; galactose metabolism; androgen and estrogen metabolism; glycine, serine, and threonine metabolism; valine, leucine, and isoleucine degradation; arginine and proline metabolism; histidine metabolism; tryptophan metabolism; d-arginine and ornithine metabolism; glycerolipid metabolism were overrepresented in library C2. Metabolic pathways of sterol, vitamin K, vitamin E, carotenoids biosynthesis; sulfur metabolism: reduction and fixation; DNA polymerase; cytochrome C oxidase were only observed in library C1, while those of fatty acid biosynthesis (path 2), styrene degradation, and Vitamine B6 metabolism were only observed in library C2.
The results showed that genes related to mycoparasitism were differentially expressed. Two different sequences similar to the 48-KDa endochitinase and 46-KDa endochitinase were identified in libraries C1 and C2, respectively. Since library C1 was obtained from cultivation on PDA medium, the 48
KDa endochitinase homolog might play a role in the dissolution and formation of the cell wall of C. cupreum
. Similarly, because library C2 was constructed under conditions associated with mycoparasitism, the 46 KDa endochitinase homolog is expected to be involved in cell wall degradation of the fungal pathogen during the mycoparasitic process.
The conditions used for construction of library C2 were aimed at in vitro
simulation of the mycoparasitic process, which is triggered by the recognition of the structural character of the pathogenic fungal cell wall. As a result, the genes involved in signal transduction pathways of mycoparasitism were acquired. Examples include homologue of gene encoding an ABC transporter (ATP-binding cassette transporter, DV548480
, ) and MAP-kinase A (Tmk1 of T. atroviride
). Four ESTs have sequence homology to an ABC transporter, it was also observed previously in other fungal pathogens (Gibberella pulicaris
and Sclerotinia sclerotiorum
) Mehrabi et al. [14
] as potential pathogenicity factors responsible for tolerance to phytoalexins or a pathogenicity factor for the host Fleissner et al. [15
] and Li et al. [16
]. Studies on signal transduction pathways from Trichoderma
strains revealed the involvement of MAP-kinases in the mycoparasitic interaction, including production of hydrolytic enzymes such as chitinases and secretion of antibiotic substances [17
DV548480 shared 73% identity with ABC transporter of Myceliophthora thermophila ATCC 42464.
In this study, we sequenced and analyzed two independent cDNA libraries, providing the first comparative analysis of the transcriptome of C. cupreum under different conditions. The findings provide an entry point for understanding further the molecular mechanisms of this fungus and will also help to advance our efforts in developing novel strategies for biocontrol of fungal diseases.