Systems biological approaches towards understanding cellulase production by Trichoderma reesei

AbstractRecent progress and improvement in “-omics” technologies has made it possible to study the physiology of organisms by integrated and genome-wide approaches. This bears the advantage that the global response, rather than isolated pathways and circuits within an organism, can be investigated (“systems biology”). The sequencing of the genome of Trichoderma reesei (teleomorph Hypocrea jecorina), a fungus that serves as a major producer of biomass-degrading enzymes for the use of renewable lignocellulosic material towards production of biofuels and biorefineries, has offered the possibility to study this organism and its enzyme production on a genome wide scale. In this review, I will highlight the use of genomics, transcriptomics, proteomics and metabolomics towards an improved and novel understanding of the biochemical processes that involve in the massive overproduction of secreted proteins

Identification of potential marker genes for <i>Trichoderma harzianum</i> strains with high antagonistic potential against <i>Rhizoctonia solani</i> by a rapid subtraction hybridization approach

A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones&#8212;acetyl-xylane esterase AXE1 and endoglucanase Cel61b&#8212;showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermediate to poor antagonistic activity. These clones are promising candidates for use as markers in the screening of improved T. harzianum biocontrol strains

The role of pheromone receptors for communication and mating in Hypocrea jecorina (Trichoderma reesei)

AbstractDiscovery of sexual development in the ascomycete Trichoderma reesei (Hypocrea jecorina) as well as detection of a novel class of peptide pheromone precursors in this fungus indicates promising insights into its physiology and lifestyle. Here we investigated the role of the two pheromone receptors HPR1 and HPR2 in the H. jecorina pheromone-system.We found that these pheromone receptors show an unexpectedly high genetic variability among H. jecorina strains. HPR1 and HPR2 confer female fertility in their cognate mating types (MAT1-1 or MAT1-2, respectively) and mediate induction of fruiting body development. One compatible pheromone precursor–pheromone receptor pair (hpr1–hpp1 or hpr2–ppg1) in mating partners was sufficient for sexual development. Additionally, pheromone receptors were essential for ascospore development, hence indicating their involvement in post-fertilisation events.Neither pheromone precursor genes nor pheromone receptor genes of H. jecorina were transcribed in a strictly mating type dependent manner, but showed enhanced expression levels in the cognate mating type. In the presence of a mating partner under conditions favoring sexual development, transcript levels of pheromone precursors were significantly increased, while those of pheromone receptor genes do not show this trend. In the female sterile T. reesei strain QM6a, transcriptional responses of pheromone precursor and pheromone receptor genes to a mating partner were clearly altered compared to the female fertile wild-type strain CBS999.97. Consequently, a delayed and inappropriate response to the mating partner may be one aspect causing female sterility in QM6a

The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

<p>Abstract</p> <p>Background</p> <p>In the biotechnological workhorse <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression.</p> <p>Results</p> <p>As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the <it>T. reesei </it>class I phosducin-like protein gene <it>phlp1 </it>and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in <it>T. reesei</it>. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency.</p> <p>Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δ<it>phlp1</it>. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δ<it>phlp1</it>. The lack of GNB1 drastically diminished ascospore discharge in <it>T. reesei</it>.</p> <p>Conclusions</p> <p>The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of <it>T. reesei</it>, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light responsiveness, glycoside hydrolase gene transcription and sexual development.</p

Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea

<p>Abstract</p> <p>Background</p> <p>Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi. Their main function is to confer hydrophobicity to fungal surfaces in contact with air or during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or themselves resulting in morphogenetic signals. Based on their hydropathy patterns and solubility characteristics, hydrophobins are divided into two classes (I and II), the latter being found only in ascomycetes.</p> <p>Results</p> <p>We have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus <it>Trichoderma/Hypocrea</it>, using three draft sequenced genomes (<it>H. jecorina = T. reesei, H. atroviridis = T. atroviride; H. virens = T. virens</it>) an additional 14,000 ESTs from six other Trichoderma spp. (<it>T. asperellum, H. lixii = T. harzianum, T. aggressivum </it>var. <it>europeae, T. longibrachiatum</it>, <it>T</it>. cf. <it>viride</it>). The former three contained six, ten and nine members, respectively. Ten is the highest number found in any ascomycete so far. All the hydrophobins we examined had the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these hydrophobins (HFBs)contained an extended N-terminus rich in either proline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades containing duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (<it>K</it>_<it>S</it>/<it>K</it>_{<it>a </it>}>> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2–4) from each species, and most were from Sordariomycetes. A combined phylogeny of these sequences with those of <it>Trichoderma </it>showed that the <it>Trichoderma </it>HFBs mostly formed their own clades, whereas those of other Sordariomycetes occurred in shared clades.</p> <p>Conclusion</p> <p>Our study shows that the genus <it>Trichoderma/Hypocrea </it>has a proliferated arsenal of class II hydrophobins which arose by birth-and-death evolution followed by purifying selection.</p

Trichoderma aureoviride: phylogenetic position and characterization

The identity of strains identified as Trichoderma aureoviride/Hypocrea aureoviridis was reconsidered. Trichoderma aureoviride was isolated originally from a specimen identified as H. aureoviridis and thus is H. aureoviridis. The morphological and molecular characters of most strains identified as T. aureoviride differ from those of the ex-type but are more typical of T. harzianum, a member of sect. Pachybasium. Molecular data do not support inclusion of T. aureoviride in sect. Trichoderma, nor was there strong phenotypic similarity between H. aureoviridis and H. rufa. In the ITS phylogeny the T. aureoviride ex-type and other collections of H. aureoviridis form a strongly supported clade that is separate from any other recognized section of Trichoderma. Hypocrea vinosa, which was originally included in the T. aureoviride aggregate species concept, is distinct from T. aureoviride, but closely allied with H. rufa/T. viride. Trichoderma aureoviride/H. aureoviridis is a rare species, restricted to the UK and the Netherlands. We redefine T. aureoviride, limiting it to strains with very slow growth rate, effuse conidiation, and the ITS-1 and 2 sequence type D.Peer Reviewe

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

<p>Abstract</p> <p>Background</p> <p>The hypercellulolytic mutant <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) RUT C30 is the <it>H. jecorina </it>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 <it>cre1 </it>and is consequently carbon catabolite derepressed. To date this and an additional frame-shift mutation in the glycoprotein-processing β-glucosidase II encoding gene are the only known genetic differences in strain RUT C30.</p> <p>Results</p> <p>In the present paper we show that <it>H. jecorina </it>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 <it>cre1 </it>locus. The mutation of the <it>cre1 </it>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 α-linked oligo- and polyglucosides (loss of maltose permease), or disturbance of osmotic homeostasis.</p> <p>Conclusion</p> <p>Our data place a general caveat on the use of <it>H. jecorina </it>RUT C30 for further basic research.</p

Identification of potential marker genes for Trichoderma harzianum strains with high antagonistic potential against Rhizoctonia solani by a rapid subtraction hybridization approach.

A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones—acetyl-xylane esterase AXE1 and endoglucanase Cel61b—showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermediate to poor antagonistic activity. These clones are promising candidates for use as markers in the screening of improved T. harzianum biocontrol strains

Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei)

<p>Abstract</p> <p>Background</p> <p>The filamentous ascomycete <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi.</p> <p>Results</p> <p>Analysis of the regulatory targets of the G-protein α subunit GNA1 in <it>H. jecorina </it>revealed a carbon source and light-dependent role in signal transduction. Deletion of <it>gna1 </it>led to significantly decreased biomass formation in darkness in submersed culture but had only minor effects on morphology and hyphal apical extension rates on solid medium. Cellulase gene transcription was abolished in Δ<it>gna1 </it>on cellulose in light and enhanced in darkness. However, analysis of strains expressing a constitutively activated GNA1 revealed that GNA1 does not transmit the essential inducing signal. Instead, it relates a modulating signal with light-dependent significance, since induction still required the presence of an inducer. We show that regulation of transcription and activity of GNA1 involves a carbon source-dependent feedback cycle. Additionally we found a function of GNA1 in hydrophobin regulation as well as effects on conidiation and tolerance of osmotic and oxidative stress.</p> <p>Conclusion</p> <p>We conclude that GNA1 transmits a signal the physiological relevance of which is dependent on both the carbon source as well as the light status. The widespread consequences of mutations in GNA1 indicate a broad function of this Gα subunit in appropriation of intracellular resources to environmental (especially nutritional) conditions.</p