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Genome-wide analysis of chitinase genes in the Hypocrea jecorina (anamorph: Trichoderma reesei) genome database revealed the presence of 18 ORFs encoding putative chitinases, all of them belonging to glycoside hydrolase family 18. Eleven of these encode yet undescribed chitinases. A systematic nomenclature for the H. jecorina chitinases is proposed, which designates the chitinases corresponding to their glycoside hydrolase family and numbers the isoenzymes according to their pI from Chi18-1 to Chi18-18. Phylogenetic analysis of H. jecorina chitinases, and those from other filamentous fungi, including hypothetical proteins of annotated fungal genome databases, showed that the fungal chitinases can be divided into three groups: groups A and B (corresponding to class V and III chitinases, respectively) also contained the so Trichoderma chitinases identified to date, whereas a novel group C comprises high molecular weight chitinases that have a domain structure similar to Kluyveromyces lactis killer toxins. Five chitinase genes, representing members of groups A–C, were cloned from the mycoparasitic species H. atroviridis (anamorph: T. atroviride). Transcription of chi18-10 (belonging to group C) and chi18-13 (belonging to a novel clade in group B) was triggered upon growth on Rhizoctonia solani cell walls, and during plate confrontation tests with the plant pathogen R. solani. Therefore, group C and the novel clade in group B may contain chitinases of potential relevance for the biocontrol properties of Trichoderma.
After cellulose, chitin is the second most abundant organic source in nature . The polymer is composed of β-(1,4)-linked units of the amino sugar N-acetylglucosamine. It is a renewable resource, extracted mainly from shellfish waste, and can be processed into many derivatives, which are used for a number of commercial products such as medical applications (e.g. surgical thread), cosmetics, dietary supplements, agriculture and water treatment [1–3].
Various organisms produce chitinolytic enzymes (EC 126.96.36.199), which hydrolyze the β-1,4-glycosidic linkage . The chitinases currently known are divided into two families (family 18 and family 19) on the basis of their amino acid sequences . These two families do not share sequence similarity and display different 3D structures: family 18 chitinases have a catalytic (α/β)8-barrel domain [6–9], while family 19 enzymes have a bilobal structure and are predominantly composed of α-helices [10–12]. They also differ in their enzymatic mechanism: family 18 chitinases have a retaining mechanism, which results in chito-oligosaccharides being in the β-anomeric configuration, whereas family 19 chitinases have an inverting mechanism and consequently the products are α-anomers. Another difference is the sensitivity to allosamidin, which inhibits only family 18 chitinases . N-acetylhexosaminidases (EC 188.8.131.52), which cleave chito-oligomers and also chitin progressively from the nonreducing end and release only N-acetylglucosamine monomers, belong to glycoside hydrolase family 20 .
Some species of the imperfect soil fungus, Trichoderma[e.g. T. harzianum (teleomorph Hypocrea lixii), T. virens (teleomorph H. virens), T. asperellum and T. atroviride (teleomorph H. atroviridis)], are potent mycoparasites of several plant pathogenic fungi that cause severe crop losses each year, and are therefore used in agriculture as biocontrol agents. Biocontrol is considered to be an attractive alternative to the strong dependence of modern agriculture on fungicides, which may cause environmental pollution and selection of resistant strains. Lysis of the host cell wall of the plant pathogenic fungi has been demonstrated to be an important step in the mycoparasitic attack [14–17]. Consequently, with chitin being a major cell wall component of plant pathogens like, for example, Rhizoctonia solani, Botrytis cinerea and Sclerotinia sclerotium, several chitinase genes have been cloned from Trichoderma spp. [18–25] and, for some, the encoded protein has also been characterized [26,27]. Recently, the chitinase, Ech30, from H. atroviridis was overexpressed in Escherichia coli and characterized , but neither its expression pattern nor its biological relevance were studied. The possible roles of the endochitinases, Ech42 and Chit33, and the N-acetylglucosaminidase, Nag1, in mycoparasitism have been investigated [29–34].
In order to obtain a comprehensive insight into the chitinolytic potential of Trichoderma, we screened the recently published genome sequence of H. jecorina (anamorph: T. reesei) for chitinase-encoding genes. In this study, we present a supposedly complete list of chitinases of Trichoderma, and demonstrate their evolutionary relationships to each other and to those from other fungi. The chitinases were characterized in silico and we propose a unifying nomenclature for the large number of chitinase-encoding genes that can be found in the H. jecorina genome. Finally, five selected chitinase genes were cloned from the mycoparasitic species H. atroviridis and their transcription studied under conditions relevant for chitinase formation and mycoparasitism. A member of a new group of high-molecular-weight chitinases (chi18-10), unidentified, to date, in filamentous fungi, thereby shows a transcription profile which suggests that it may be relevant for biocontrol.
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In this study we identified 18 genes encoding proteins belonging to glycoside hydrolase family 18 and two members of family 20 in the H. jecorina genome, whereas no members of family 19, primarily found in plants, were detected. Previously, most authors named Trichoderma chitinases according to the putative Mr, thereby frequently also attaching an abbreviation of the species from which it was cloned [23,25,35]. However, the large number of chitinases in H. jecorina presented in this study, and the clear presence of orthologues in other filamentous fungi, makes a more systematic nomenclature for these proteins necessary. In this article we have therefore applied the rules of the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) to the Trichoderma chitinases, and numbered the isoenzymes starting with the protein having the lowest theoretical pI . As we assume that we have assessed the complete chitinase spectrum of H. jecorina, we propose that the names of Trichoderma chitinases should be based on their H. jecorina orthologue and then be numbered accordingly. In addition, we follow the proposal of Henrissat , to include the glycoside hydrolase family identification number after the three letter code of the gene (chi). Chi was chosen because it is already the most commonly used name for chitinases from other organisms.
Seventeen of the H. jecorina family 18 chitinases members could be classified into three phylogenetic groups also containing several chitinases from other filamentous fungi, whereas Chi18-15 could not be aligned with any of them. Chi18-15 was previously cloned from T. asperellum and characterized, by Viterbo et al., as Chit36 [24,25]. The only orthologues that could be found in other organisms are a chitinase from the entomopathogen C. bassiana, which has been demonstrated to be involved in the attack of the fungus on insects  and two chitinases from Streptomyces spp. These data suggest that the occurrence of chi18-15 in the genome of H. jecorina, H. atroviridis and C. bassiana is caused by horizontal transfer, which – because C. bassiana and Trichoderma are both members of the Hypocreaceae– has apparently taken place rather recently (110–150 million years ago) .
All other family 18 chitinases have orthologues in filamentous fungi, including the phylogenetically diverse ascomycetes A. nidulans, N. crassa and G. zeae. This indicates that the ancestors of these genes/proteins were formed very early during the evolution of ascomycetes and their gene products therefore very likely fulfil vital functions in the fungal life cycle and/or ecology.
Particularly for chitinases of group A, orthologues were found in almost all other filamentous fungi. The closest neighbours to Trichoderma chitinases were mostly the G. zeae orthologues, indicating that evolution of these genes parallels the evolution of these species. In fact, one of these genes, chi18-5 (ech42), is used as a locus for phylogenetic analysis of the genus Trichoderma[80,81]. Chi18-5 is a chitinase that is well conserved throughout the ascomycetes, and is therefore likely to have a vital function in them. This is supported by the finding that for H. jecorina chi18-5, and the closely related chi18-7, encoding a putatively intracellular chitinase, a large number of ESTs can be found in the H. jecorina genome database, whereas none, or only two to four ESTs, were sequenced from other chitinases. It is intriguing that this gene has also been frequently investigated with respect to its involvement in mycoparasitism and biocontrol by H. atroviridis, H. lixii and H. virens[29,33,34,73,82]. Knockouts of this gene resulted in some, albeit small, reduction in biocontrol of the corresponding strains [29,34], consistent with the interpretation that chi18-5 has a rather different function in Trichoderma. As transcription of chi18-5 is triggered by carbon starvation, Brunner et al.  speculated that its main function may be associated with mycelial autolysis.
In contrast, group B, which contains chitinases with similarity to Chi18-12 (Chit33), seems to contain proteins with more species-specific functions. One striking feature of this cluster is that we could not detect any orthologue of these proteins in G. zeae, indicating that this group of chitinases is dispensable for a plant pathogenic fungus and therefore probably not essential. With the exception of Chi18-12, all members of this cluster have a fungal cellulose-binding domain (CBD) (InterPro acc. no.: IPR000254), consisting of four strictly conserved aromatic amino acid residues that are implicated in the interaction with cellulose, and four strictly conserved cysteine residues that are predicted to form two disulfide bonds . CBDs occur not only as domains of cellulose-degrading enzymes, but have also been identified in other polysaccharide-degrading enzymes (listed as CBM 1 entries in the CAZy database; http://afmb.cnrs-mrs.fr/CAZY/) . Limon et al.  demonstrated that the addition of a CBD to H. lixii Chit42 (Chi18-5) increased its activity towards high molecular mass insoluble chitin substrates, such as those found in fungal cell walls. It is therefore likely that the presence of CBDs in this cluster of family 18 chitinases may support them in chitin degradation during the mycoparasitic attack.
Interestingly, Kim et al.  reported that the CBD with highest similarity to Chi18-17 (Tv-cht1) was found in an endochitinase from the entomopathogenic fungus M. anisopliae var. acridum (CHI2; GenBank acc. no.: CAC07216). While this was true for the limited sample of chitinases available for the study, we found three chitinases from H. jecorina that are phylogenetically more close to CHI2, and indeed – together with a second chitinase from M. anisopliae (CHIT30; GenBank acc no.: AAS55554) – form a separate clade within group B. The absence of orthologous members of this clade from all other ascomycetous genomes makes it highly likely that these proteins have a special function in chitin degradation by mycoparasitic fungi (like Trichoderma) and entomopathogens (like Metarhizium). Consistent with this assumption, we showed that one member of this cluster (chi18-13) is strongly up-regulated in H. atroviridis in the presence of R. solani cell walls and in plate confrontations before contact. Thus, chi18-13, and probably also chi18-14 and chi18-16, are genes that are potentially involved in mycoparasitism and biocontrol.
It should be noted that groups A and B in the phylogenetic analysis correspond to the family 18 chitinase subgroup classes V and III, respectively. Together with the chitinase classes I, II and IV, which contain members of glycoside hydrolase family 19, this classification was used for plant chitinases prior to the glycoside hydrolase family classification [10,85]. This prompted authors to use names like fungal/plant (class III) and fungal/bacterial (class V) chitinases for these subclasses owing to similarities to either plant chitinases or bacterial chitinases [54,86]. As we detected a third subgroup of glycoside hydrolase family 18 chitinases, but our phylogenetic analysis was restricted to filamentous fungi, we simply called the subgroups (according to the clusters in Figs 2–4) group A (which is consistent with class V, also called fungal/bacterial chitinases), group B (consistent with class III and fungal/plant chitinases) and group C (a novel group of family 18 chitinases).
This third cluster (group C) of chitinases probably contains the most intriguing members of family 18. First, none of these proteins has as yet been characterized from any filamentous fungus, the cluster comprising – with the exception of A. fumigatus Chi100, for which, however, only a GenBank entry is available – only putative proteins from other fungal genome databases. Second, all of its members have a domain structure consisting of a class I chitin-binding domain (InterPro acc. no.: IPR001002; CBM 18 according to the CAZy classification) , comprising eight disulfide-linked cysteines  accompanied by two LysM domains and then followed by the glycoside family 18 domain. Although the occurrence of orthologues of these proteins in other nonmycoparasitic ascomycetes indicates that these proteins have not specifically evolved for antagonism of other fungi by Trichoderma, it is intriguing to note that these high molecular weight chitinases have high similarity to the killer toxins of certain yeasts , and chi18-10 of H. atroviridis is only expressed during growth on fungal cell walls and during plate confrontation assays, and not upon carbon starvation or growth on chitin. No protein with similarity to the γ-subunit of the yeast killer toxins – which is the actual toxicity factor – has been found in the H. jecorina genome. However, as the γ-subunit causes cell cycle arrest in yeast, it is probably dispensable for the antagonization of multicellular fungi. Rather, we speculate that Trichoderma uses a killer-toxin like mechanism to enable the penetration of antifungal molecules into its host. For this reason, we also consider this group of chitinases potentially interesting candidates for proteins that are connected with the biocontrol properties of Trichoderma.
Transcription analysis of the novel H. atroviridis chitinases chit18-2, chi18-3, chi18-4, chi18-10 and chi18-13 showed that, although transcript levels were generally rather low as they could not be detected by northern analysis and one has to be careful with interpreting the RT-PCR data quantitatively, a clear influence of different growth conditions and carbon sources could be detected. This indicates the functional diversity of the Trichoderma chitinases and that they are not just substitutes for each other, but that they indeed have specific roles in the organism. In particular, the transcript patterns of chi18-10 and chi18-13 were explicitly linked to the presence of components apparently present in the cell wall of R. solani. No striking similarities in the upstream regions of chi18-10 and chi18-13 were detected. The extensive in silico analysis of the novel H. atroviridis chitinase genes (Fig. 5) gives some hints as to which regulatory mechanisms might be important for the respective chitinase genes, but detailed promotor studies are certainly necessary to elucidate any common consensus sites and transcription factors responsible for the regulation of Trichoderma chitinases.
In this study, we showed, for the first time, that post-transcriptional regulation is involved in chitinase expression. We demonstrated that, at least for chi18-3 and chi18-13, different mRNA species were present and that their occurrence was influenced by the growth conditions. Additionally we found a Puf-binding site in the 3′-UTR of chi18-13. It should be noted that proteins with Puf RNA-binding domains (InterPro acc. no.: IPR001313) are indeed present in the H. jecorina genome. The aspect of post-transcriptional regulation has not yet been studied great detail in filamentous fungi. It comprises interesting insights into the actual protein levels that can be observed in vivo and could contribute to a more accurate understanding of enzyme-mediated events, such as mycoparasitism.