Sexual development in the industrial workhorse Trichoderma reesei.
ABSTRACT: 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.
Project description:BACKGROUND:Filamentous fungi are frequently used as production platforms in industrial biotechnology. Most of the strains involved were known as reproducing exclusively asexually thereby preventing the application of conventional strain breeding techniques. In the last decade, evidence was obtained that a number of these imperfect fungi possess a sexual life cycle, too. Trichoderma reesei, an industrial producer of enzymes for food, feed and biorefinery purposes, is heterothallic and takes a special position among industrially utilized species as all industrial strains are derived from the single MAT1-2 isolate QM6a. Consequently, strain improvement by crossing is not feasible within this strain line as this necessitates a MAT1-1 mating partner. Simply switching the mating type in one of the mating partners to MAT1-1, however, is not sufficient to produce a genotype capable of sexual reproduction with QM6a MAT1-2. RESULTS:We have used a systems biology approach to identify genes restoring sexual reproduction in the QM6a strain line. To this end, T. reesei QM6a was crossed with the MAT1-1 wild-type strain CBS999.97. The descendants were backcrossed 8-times in two lineages with QM6a to obtain mating competent MAT1-1 strains with a minimal set of CBS999.97 specific genes. Comparative genome analysis identified a total of 73 genes of which two-encoding an unknown C2H2/ankyrin protein and a homolog of the WD-protein HAM5-were identified to be essential for fruiting body formation. The introduction of a functional ham5 allele in a mating type switched T. reesei QM6a allowed sexual crossing with the parental strain QM6a. CONCLUSION:The finding that Trichoderma reesei is generally capable of undergoing sexual reproduction even under laboratory conditions raised hope for the applicability of classical breeding techniques with this fungus as known for plants and certain yeasts. The discovery that the wild-type isolate QM6a was female sterile, however, precluded any progress along that line. With the discovery of the genetic cause of female sterility and the creation of an engineered fertile strain we now provide the basis to establish sexual crossing in this fungus and herald a new era of strain improvement in T. reesei.
Project description:Discovery 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.
Project description:The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.
Project description:Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type-dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1-2 mating type and for the mating type gene mat1-2-1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T.?reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.
Project description:Background:Trichoderma reesei is one of the most frequently used filamentous fungi in industry for production of homologous and heterologous proteins. The ability to use sexual crossing in this fungus was discovered several years ago and opens up new perspectives for industrial strain improvement and investigation of gene regulation. Results:Here we investigated the female sterile strain QM6a in comparison to the fertile isolate CBS999.97 and backcrossed derivatives of QM6a, which have regained fertility (FF1 and FF2 strains) in both mating types under conditions of sexual development. We found considerable differences in gene regulation between strains with the CBS999.97 genetic background and the QM6a background. Regulation patterns of QM6a largely clustered with the backcrossed FF1 and FF2 strains. Differential regulation between QM6a and FF1/FF2 as well as clustering of QM6a patterns with those of CBS999.97 strains was also observed. Consistent mating type dependent regulation was limited to mating type genes and those involved in pheromone response, but included also nta1 encoding a putative N-terminal amidase previously not associated with development. Comparison of female sterile QM6a with female fertile strains showed differential expression in genes encoding several transcription factors, metabolic genes and genes involved in secondary metabolism. Conclusions:Evaluation of the functions of genes specifically regulated under conditions of sexual development and of genes with highest levels of transcripts under these conditions indicated a relevance of secondary metabolism for sexual development in T. reesei. Among others, the biosynthetic genes of the recently characterized SOR cluster are in this gene group. However, these genes are not essential for sexual development, but rather have a function in protection and defence against competitors during reproduction.
Project description:The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1-2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.
Project description:Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.
Project description:Due to its capability to secrete large quantities of plant biomass degrading enzymes (PBDE), Trichoderma reesei is widely applied for industrial purposes. In nature, expression of PBDE is efficiently regulated in this fungus. Several factors involved in this regulatory network have been identified. However, most of them are transcription factors. Long noncoding RNAs (lncRNAs) emerged as common players acting on epigenetic or transcriptional regulation in several eukaryotic organisms. To date, no lncRNA has been described in filamentous fungi.A lncRNA termed HAX1 was identified in T. reesei QM9414. In this study, it was characterized and evidence for its regulatory impact on cellulase expression was provided. Interestingly, different versions of HAX1 were identified in different strains (namely, QM6a, QM9414, and Rut-C30), varying in terms of RNA length. Remarkably, considerable longer variants of this lncRNA are present in hypercellulolytic strains compared to the wild-type strain QM6a. Based on these results, a correlation between RNA length and the functional impact of HAX1 on PBDE expression was supposed. This assumption was verified by overexpressing the most abundant HAX1 versions identified in QM6a, QM9414, and Rut-C30. Such HAX1 overexpression on the one hand was suitable for regaining the function in hax1 disruption strains, and on the other hand resulted in notably higher cellulase activities in QM6a, especially by the expression of longer HAX1 versions.With HAX1, for the first time the regulatory role of a lncRNA in filamentous fungi was uncovered. Besides this, a new player involved in the complex regulation of PBDE expression in T. reesei was identified. Due to its enhancing effect on cellulase activity, HAX1 was shown to be not only interesting for basic research, but also a promising candidate for expanding the set of biotechnological tools for industrial application of T. reesei.
Project description:Sexual development is regulated by a complex regulatory mechanism in fungi. For Trichoderma reesei, the light response pathway was shown to impact sexual development, particularly through the photoreceptor ENVOY. Moreover, T. reesei communicates chemically with a potential mating partner in its vicinity, a response which is mediated by the velvet family protein VEL1 and its impact on secondary metabolism. We therefore studied the regulatory interactions of ENV1 and VEL1 with a focus on sexual development. Although individual mutants in both genes are female sterile under standard crossing conditions (light-dark cycles), an altered light regime enabled sexual development, which we found to be due to conditional female sterility of ?env1, but not ?vel1. Phenotypes of growth and asexual sporulation as well as regulation of the peptide pheromone precursors of double mutants suggested that ENV1 and VEL1 balance positive and negative regulators of these functions. Additionally, VEL1 contributed to the strong deregulation of the pheromone system observed in env1 mutants. Female sterility of ?vel1 was rescued by deletion of env1 in darkness in MAT1-1, indicating a block of sexual development by ENV1 in darkness that is balanced by VEL1 in the wild-type. We conclude that ENV1 and VEL1 exert complementing functions in development of T. reesei. Our results further showed that the different developmental phenotypes of vel1/veA mutants in T. reesei and Aspergillus nidulans are not due to the presence or function of ENV1 in the VELVET regulatory pathway in T. reesei.
Project description: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.