Evolution and ecophysiology of the industrial producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a new sympatric agamospecies related to it.

BACKGROUND: Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina. METHODOLOGY/PRINCIPAL FINDINGS: We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov. CONCLUSIONS: Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour--T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies.

Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey.

BACKGROUND: Combating the action of plant pathogenic microorganisms by mycoparasitic fungi has been announced as an attractive biological alternative to the use of chemical fungicides since two decades. The fungal genus Trichoderma includes a high number of taxa which are able to recognize, combat and finally besiege and kill their prey. Only fragments of the biochemical processes related to this ability have been uncovered so far, however. RESULTS: We analyzed genome-wide gene expression changes during the begin of physical contact between Trichoderma atroviride and two plant pathogens Botrytis cinerea and Rhizoctonia solani, and compared with gene expression patterns of mycelial and conidiating cultures, respectively. About 3000 ESTs, representing about 900 genes, were obtained from each of these three growth conditions. 66 genes, represented by 442 ESTs, were specifically and significantly overexpressed during onset of mycoparasitism, and the expression of a subset thereof was verified by expression analysis. The upregulated genes comprised 18 KOG groups, but were most abundant from the groups representing posttranslational processing, and amino acid metabolism, and included components of the stress response, reaction to nitrogen shortage, signal transduction and lipid catabolism. Metabolic network analysis confirmed the upregulation of the genes for amino acid biosynthesis and of those involved in the catabolism of lipids and aminosugars. CONCLUSION: The analysis of the genes overexpressed during the onset of mycoparasitism in T. atroviride has revealed that the fungus reacts to this condition with several previously undetected physiological reactions. These data enable a new and more comprehensive interpretation of the physiology of mycoparasitism, and will aid in the selection of traits for improvement of biocontrol strains by recombinant techniques.

Sexual development in the industrial workhorse Trichoderma reesei.

Filamentous fungi are indispensable biotechnological tools for the production of organic chemicals, enzymes, and antibiotics. Most of the strains used for industrial applications have been--and still are--screened and improved by classical mutagenesis. Sexual crossing approaches would yield considerable advantages for research and industrial strain improvement, but interestingly, industrially applied filamentous fungal species have so far been considered to be largely asexual. This is also true for the ascomycete Trichoderma reesei (anamorph of Hypocrea jecorina), which is used for production of cellulolytic and hemicellulolytic enzymes. In this study, we report that T. reesei QM6a has a MAT1-2 mating type locus, and the identification of its respective mating type counterpart, MAT1-1, in natural isolates of H. jecorina, thus proving that this is a heterothallic species. After being considered asexual since its discovery more than 50 years ago, we were now able to induce sexual reproduction of T. reesei QM6a and obtained fertilized stromata and mature ascospores. This sexual crossing approach therefore opens up perspectives for biotechnologically important fungi. Our findings provide a tool for fast and efficient industrial strain improvement in T. reesei, thus boosting research toward economically feasible biofuel production. In addition, knowledge of MAT-loci and sexual crossing techniques will facilitate research with other Trichoderma spp. relevant for agriculture and human health.

The beta-N-acetylglucosaminidases NAG1 and NAG2 are essential for growth of Trichoderma atroviride on chitin.

The chitinolytic enzyme machinery of fungi consists of chitinases and beta-N-acetylglucosaminidases. These enzymes are important during the fungal life cycle for degradation of exogenous chitin, which is the second most abundant biopolymer, as well as fungal cell-wall remodelling. In addition, involvement of chitinolytic enzymes in the lysis of the host cell wall in mycoparasitic Trichoderma spp. has been reported. In view of the fact that fungi have on average 15-20 chitinases, but only two beta-N-acetylglucosaminidases, the question arises how important the latter enzymes actually are for various aspects of chitin degradation. In this study, the role of two beta-N-acetylglucosaminidases, NAG1 and NAG2, was analysed in the mycoparasitic fungus Trichoderma atroviride. No beta-N-acetylglucosaminidase activity was detected in T. atrovirideDeltanag1Deltanag2 strains, suggesting that NAG1 and NAG2 are the only enzymes in T. atroviride that possess this activity. Deltanag1Deltanag2 strains were not able to grow on chitin and chitobiose, but the presence of either NAG1 or NAG2 was sufficient to restore growth on chitinous carbon sources in solid media. Our results demonstrated that T. atroviride cannot metabolize chitobiose but only the monomer N-acetylglucosamine, and that N-acetylglucosaminidases are therefore essential for the use of chitin as a nutrient source. NAG1 is predominantly secreted into the medium, whereas NAG2 mainly remains attached to the cell wall. No physiological changes or reduction of the mycoparasitic potential of T. atroviride was detected in the double knockout strains, suggesting that the use of chitin as carbon source is only of minor importance for these processes.

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism.

The Hypocrea jecorina LXR1 was described as the first fungal L-xylulose reductase responsible for NADPH dependent reduction of L-xylulose to xylitol in L-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal D-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus niger mtdA are not induced by L-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on L-arabinose and L-xylulose reductase activity remains unaltered whereas D-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a D-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.

Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.

The industrially applied ascomycete Hypocrea jecorina (synonym: Trichoderma reesei) exhibits a low rate of exogenous DNA integration by homologous recombination (HR). This hinders the high-throughput generation of strains by gene replacement and is therefore impeding systematic functional gene analyses towards, e.g. strain improvement for protein or enzyme production. To increase the rate of HR events during fungal transformation we identified and deleted the orthologue of the human KU70 in H. jecorina, which is required for the nonhomologous end joining (NHEJ) pathway and responsible for ectopic DNA integration. The effect of the absence of the H. jecorina tku70 on gene targeting was tested by deletion of two so far uncharacterized genes encoding a short chain dehydrogenase and a fungal specific transcription factor. Efficiency of gene targeting for both genes was >95% in a Deltatku70 strain when 1kb homologous flanking regions were used in the deletion construct. This is a significant increase in targeting efficiency compared to the parental - non-tku70 deleted - strain TU-6 where a gene knock-out frequency of only 5-10% was observed. Together with the recently annotated genomic sequence of H. jecorina, this system provides a useful tool for a genome-wide functional gene analysis on a high-throughput scale to improve the biotechnological potential of this fungus.

The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome.

BACKGROUND: The hypercellulolytic mutant Hypocrea jecorina (anamorph Trichoderma reesei) RUT C30 is the H. jecorina strain most frequently used for cellulase fermentations and has also often been employed for basic research on cellulase regulation. This strain has been reported to contain a truncated carbon catabolite repressor gene cre1 and is consequently carbon catabolite derepressed. To date this and an additional frame-shift mutation in the glycoprotein-processing beta-glucosidase II encoding gene are the only known genetic differences in strain RUT C30. RESULTS: In the present paper we show that H. jecorina RUT C30 lacks an 85 kb genomic fragment, and consequently misses additional 29 genes comprising transcription factors, enzymes of the primary metabolism and transport proteins. This loss is already present in the ancestor of RUT C30--NG 14--and seems to have occurred in a palindromic AT-rich repeat (PATRR) typically inducing chromosomal translocations, and is not linked to the cre1 locus. The mutation of the cre1 locus has specifically occurred in RUT C30. Some of the genes that are lacking in RUT C30 could be correlated with pronounced alterations in its phenotype, such as poor growth on alpha-linked oligo- and polyglucosides (loss of maltose permease), or disturbance of osmotic homeostasis. CONCLUSION: Our data place a general caveat on the use of H. jecorina RUT C30 for further basic research.

Application of DNA bar codes for screening of industrially important fungi: the haplotype of Trichoderma harzianum sensu stricto indicates superior chitinase formation.

Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.

Epl1, the major secreted protein of Hypocrea atroviridis on glucose, is a member of a strongly conserved protein family comprising plant defense response elicitors.

We used a proteomic approach to identify constitutively formed extracellular proteins of Hypocrea atroviridis (Trichoderma atroviride), a known biocontrol agent. The fungus was cultivated on glucose and the secretome was examined by two-dimensional gel electrophoresis. The two predominant spots were identified by MALDI MS utilizing peptide mass fingerprints and amino acid sequence tags obtained by postsource decay and/or high-energy collision-induced dissociation (MS/MS) experiments, and turned out to be the same protein (12 629 Da as determined with MS, pI 5.5-5.7), probably representing the monomer and the dimer. The corresponding gene was subsequently cloned from H. atroviridis and named epl1 (eliciting plant response-like), because it encodes a protein that exhibits high similarity to the cerato-platanin family, which comprises proteins such as cerato-platanin from Ceratocystis fimbriata f. sp. platani and Snodprot1 of Phaeosphaeria nodorum, which have been reported to be involved in plant pathogenesis and elicitation of plant defense responses. Additionally, based on the similarity of the N-terminus to that of H. atroviridis Epl1, we conclude that a previously identified 18 kDa plant response elicitor isolated from T. virens is an ortholog of epl1. Our results showed that epl1 transcript was present under all growth conditions tested, which included the carbon sources glucose, glycerol, l-arabinose, d-xylose, colloidal chitin and cell walls of the plant pathogen Rhizoctonia solani, and also plate confrontation assays with R. solani. Epl1 transcript could even be detected under osmotic stress, and carbon and nitrogen starvation.

A screening system for carbon sources enhancing beta-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride).

To identify carbon sources that trigger beta-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on alpha-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Deltanag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Deltanag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression.

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.

A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases.

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.

The G protein alpha subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites.

Trichoderma mycoparasitism includes recognition, attack, overgrowth and lysis of the host fungus accompanied by morphological changes and secretion of hydrolytic enzymes and antibiotics. Studying the underlying signal transduction pathways, the tga1 gene encoding a Galpha subunit of Trichoderma atroviride P1 was analysed. A Deltatga1 mutant showed continuous sporulation and elevated internal steady-state cAMP levels. tga1 gene deletion resulted in a complete loss of mycoparasitic overgrowth and lysis of Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum during direct confrontation, although infection structure formation was unaffected. The reduced mycoparasitic abilities were reflected by strongly decreased chitinase activities and reduced nag1 and ech42 gene transcription. Furthermore, production of 6-pentyl-alpha-pyrone and of metabolites with sesquiterpene structure was reduced in the Deltatga1 mutant. Regardless of these deficiencies, the mutant displayed an enhanced growth inhibition of the host fungi by over-producing other low molecular weight antifungal metabolites, suggesting opposite roles of Tga1 in regulating the biosynthesis of different antifungal substances in T. atroviride.

The fungal STRE-element-binding protein Seb1 is involved but not essential for glycerol dehydrogenase (gld1) gene expression and glycerol accumulation in Trichoderma atroviride during osmotic stress.

Fungi counteract extracellular osmotic pressure by producing intracellular polyols to prevent loss of water. In yeast osmotic signaling involves a MAP-kinase pathway culminating at the STRE-binding transcription factors Msn2/4. We investigated the role of a putative STRE-binding orthologue of Trichoderma atroviride, Seb1, in osmotic stress signaling. T. atroviride, subjected to osmotic stress (10% glucose or glycerol, 1M KCl or NaCl), responds by raising its intracellular glycerol level. In contrast to Aspergillus nidulans, no erythritol is accumulated. Accumulation of glycerol levels under osmotic stress is strongly reduced in a seb1 deletion strain. To investigate glycerol biosynthesis in T. atroviride, the genes encoding glycerol dehydrogenase (gld1) and glycerol-3-phosphate dehydrogenase (gfd1) were cloned and characterized. Although both genes contain STRE-elements in their 5'-non-coding regions, only gld1 mRNA accumulates in response to osmotic stress, whereas expression of gfd1 remains at a constitutive level. In comparison to A. nidulans gld1 transcript levels in T. atroviride rise very slowly under conditions of salt stress. Deletion of seb1 results in a delayed accumulation of the gld1 transcript, but final levels match those in the wild-type whereas gfd1 transcript accumulation remains unaffected. Assays for glycerol dehydrogenase and glycerol-3-phosphate dehydrogenase enzymatic activities reveal an increase of the former--whereas the latter remains mainly unaffected--in the wild-type and the Deltaseb1 strain under different kinds of osmotic stress. The data suggest that Seb1 is only involved in, but not essential for osmotic stress response which is in contrast to the yeast orthologues Msn2/4.