The information highways of a biotechnological workhorse – signal transduction in Hypocrea jecorina

<p>Abstract</p> <p>Background</p> <p>The ascomycete <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) is one of the most prolific producers of biomass-degrading enzymes and frequently termed an industrial workhorse. To compete for nutrients in its habitat despite its shortcoming in certain degradative enzymes, efficient perception and interpretation of environmental signals is indispensable. A better understanding of these signals as well as their transmission machinery can provide sources for improvement of biotechnological processes.</p> <p>Results</p> <p>The genome of <it>H. jecorina </it>was analysed for the presence and composition of common signal transduction pathways including heterotrimeric G-protein cascades, cAMP signaling, mitogen activated protein kinases, two component phosphorelay systems, proteins involved in circadian rhythmicity and light response, calcium signaling and the superfamily of Ras small GTPases. The results of this survey are discussed in the context of current knowledge in order to assess putative functions as well as potential impact of alterations of the respective pathways.</p> <p>Conclusion</p> <p>Important findings include an additional, bacterial type phospholipase C protein and an additional 6-4 photolyase. Moreover the presence of 4 RGS-(Regulator of G-protein Signaling) proteins and 3 GprK-type G-protein coupled receptors comprising an RGS-domain suggest a more complex posttranslational regulation of G-protein signaling than in other ascomycetes. Also the finding, that <it>H. jecorina</it>, unlike yeast possesses class I phosducins which are involved in phototransduction in mammals warrants further investigation. An alteration in the regulation of circadian rhythmicity may be deduced from the extension of both the class I and II of casein kinases, homologues of which are implicated in phosphorylation of FRQ in <it>Neurospora crassa</it>. On the other hand, a shortage in the number of the pathogenicity related PTH11-type G-protein coupled receptors (GPCRs) as well as a lack of microbial opsins was detected. Considering its efficient enzyme system for breakdown of cellulosic materials, it came as a surprise that <it>H. jecorina </it>does not possess a carbon sensing GPCR.</p

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

Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei)

<p>Abstract</p> <p>Background</p> <p>Sulphur compounds like cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. In the filamentous fungus <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) little is known about the participants in this circuit.</p> <p>Results</p> <p>Analyses of proteins binding to the cellulase activating element (CAE) within the promotor of the cellobiohydrolase <it>cbh2 </it>gene led to the identification of a putative E3 ubiquitin ligase protein named LIMPET (LIM1), which is an orthologue of the sulphur regulators SCON-2 of <it>Neurospora crassa </it>and Met30p of <it>Saccharomyces cerevisiae</it>. Transcription of <it>lim1 </it>is specifically up-regulated upon sulphur limitation and responds to cellulase inducing conditions. In addition, light dependent stimulation/shut down of cellulase gene transcription by methionine in the presence of sulphate was observed. Further, <it>lim1 </it>transcriptionally reacts to a switch from constant darkness to constant light and is subject to regulation by the light regulatory protein ENVOY. Thus <it>lim1</it>, despite its function in sulphur metabolite repression, responds both to light as well as sulphur- and carbon source. Upon growth on cellulose, the uptake of sulphate is dependent on the light status and essential for growth in light. Unlike other fungi, growth of <it>H. jecorina </it>is not inhibited by selenate under low sulphur conditions, suggesting altered regulation of sulphur metabolism. Phylogenetic analysis of the five sulphate permeases found in the genome of <it>H. jecorina </it>revealed that the predominantly mycelial sulphate permease is lacking, thus supporting this hypothesis.</p> <p>Conclusion</p> <p>Our data indicate that the significance of the sulphate/methionine-related signal with respect to cellulase gene expression is dependent on the light status and reaches beyond detection of sulphur availability.</p

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

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

Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa

<p>Abstract</p> <p>Background</p> <p>Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. <it>Neurospora crassa </it>has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in <it>N. crassa</it>.</p> <p>Results</p> <p>We show that the <it>N. crassa </it>photoreceptor genes <it>wc-1, wc-2 </it>and <it>vvd </it>are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of <it>vvd </it>expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose.</p> <p>Conclusions</p> <p>Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the <it>wc-1 </it>and <it>wc-2 </it>mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein <it>cpc-1</it>, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in <it>N. crassa</it>.</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

Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process

<p>Abstract</p> <p>Background</p> <p>In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus <it>Hypocrea jecorina </it>(anamorph <it>Trichoderma reesei</it>) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function.</p> <p>Results</p> <p>The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness.</p> <p>Conclusion</p> <p>The different responses indicate that light impacts fungi like <it>H. jecorina </it>at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light.</p

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