Light, stress, sex and carbon - The photoreceptor ENVOY as a central checkpoint in the physiology of Trichoderma reesei.

Trichoderma reesei represents one of the most prolific producers of homologous and heterologous proteins. Discovery of the photoreceptor ENV1 as a regulator of cellulase gene expression initiated analysis of light response pathways and their physiological relevance for T. reesei. The function of ENV1 in regulation of plant cell wall degrading enzymes is conserved in Neurospora crassa. ENV1 emerged as a central checkpoint for integration of nutrient sensing, light response and development. This photoreceptor exerts its function by influencing transcript abundance and feedback cycles of the alpha subunits of the heterotrimeric G-protein pathway and impacts regulation of the beta and gamma subunits via mutual regulation with the phosducin PhLP1. The output of regulation by ENV1 is in part mediated by the cAMP pathway and likely aimed at cellulose recognition. Lack of ENV1 causes deregulation of the pheromone system and female sterility in light. A regulatory interconnection with VEL1 and influence on other regulators of secondary metabolism like YPR2 as well as polyketide synthase encoding genes indicates a function in secondary metabolism. The function of ENV1 in integrating light response with signaling of osmotic and oxidative stress is evolutionary conserved in Hypocreales and distinct from other sordariomycetes including N. crassa.

The mating type locus protein MAT1-2-1 of Trichoderma reesei interacts with Xyr1 and regulates cellulase gene expression in response to light.

Cellulase production in the model cellulolytic fungus Trichoderma reesei is subject to a variety of environmental and physiological conditions involving an intricate regulatory network with multiple transcription factors. Here, we identified the mating type locus protein MAT1-2-1 as an interacting partner for the key transcriptional activator Xyr1 of T. reesei cellulase genes. Yeast two-hybrid and GST pulldown analyses revealed that MAT1-2-1 directly interacted with the putative transcription activation domain (AD, 767~940 aa) and the middle homology region (MHR2, 314~632 aa) of Xyr1. Disruption of the mat1-2-1 gene compromised the induced expression of cellulase genes with Avicel in response to light or with lactose. Chromatin immunoprecipitation (ChIP) demonstrated that MAT1-2-1 was recruited to the cbh1 (cellobiohydrolase 1-encoding) gene promoter in a Xyr1-dependent manner. These results strongly support an important role of MAT1-2-1 as a physiological cofactor of Xyr1, and suggest that MAT1-2-1 represents another regulatory node that integrates the light response with carbon source signaling to fine tune cellulase gene transcription.

A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei.

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. SIGNIFICANCE AND IMPACT OF THE STUDY: There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future.

Microelectric Current Treatment Enhanced Biodegradation of Pumpkin Lignocelluloses by Trichoderma reesei RUT-C30.

A homemade microcurrent reactor was used to treat the fermentation of Trichoderma reesei. Results indicated that the yield of saccharides for T. reesei RUT-C30 cultivated in pumpkin lignocellulose broth reaches 38.86% (w/w) when a microcurrent treatment (20 mA, at the 48th hour for 60 min) was carried out, which is significantly higher than the control group (p < 0.05). Additionally, activities of endoglucanase, cellobiohydrolase, xylanase, and pectinase were significantly increased in days 3-7. Furthermore, the fungal growth was facilitated by microelectric treatment, showing a 0.57-fold increase of spore numbers at the sixth day of cultivation. Besides, the monosaccharide composition, including glucose (1.03 mg/mL), xylose (0.12 mg/mL), arabinose (0.31 mg/mL), and fructose (0.13 mg/mL), extracted from the reactor was higher than that without the current treatment. In this work, we improved the biodegradation of lignocellulosic wastes by applying a microcurrent to lignocellulose-degrading fungal cultures and provided a new idea for the lignocellulose material pretreatment and bioconversion.

Interrelationships of VEL1 and ENV1 in light response and development in Trichoderma reesei.

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 Δenv1, but not Δvel1. 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 Δvel1 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.

Strain improvement of Trichoderma viride for increased cellulase production by irradiation of electron and (12)C(6+)-ion beams.

OBJECTIVES: To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a (12)C(6+)-ion beam. RESULTS: Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity. CONCLUSIONS: Mutagenesis with electron and (12)C(6+)-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

The Trichoderma atroviride cryptochrome/photolyase genes regulate the expression of blr1-independent genes both in red and blue light.

Quantitative transcriptome analysis led to the identification of 331 transcripts regulated by white light. Evaluation of the response to white light in mutants affected in the previously characterized blue-light receptor Blr1, demonstrated the existence of both Blr1-dependent and independent responses. Functional categorization of the light responsive genes indicated the effect of light on regulation of various transcription factors, regulators of chromatin structure, signaling pathways, genes related to different kinds of stress, metabolism, redox adjustment, and cell cycle among others. In order to establish the participation of other photoreceptors, gene expression was validated in response to different wavelengths. Gene regulation by blue and red light suggests the involvement of several photoreceptors in integrating light signals of different wavelengths in Trichoderma atroviride. Functional analysis of potential blue light photoreceptors suggests that several perception systems for different wavelengths are involved in the response to light. Deletion of cry1, one of the potential photoreceptors, resulted in severe reduction in the photoreactivation capacity of the fungus, as well as a change in gene expression under blue and red light.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

Effects of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride.

AIMS: The goal was to determine the effect of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride LU132 against Rhizoctonia solani. METHODS AND RESULTS: The incubation temperatures of 20, 25 or 30°C were assessed on the production of T. atroviride conidia under constant light over a 25 and 50 days periods. The resulting conidia were also studied for germination and bioactivity. Conidium production was maximum at 25°C after 20 days. The second peak of conidium production occurred at 45-50 days. Incubation at 25°C after 15 days showed optimum production of T. atroviride LU132. Conidia produced at 30°C gave the greatest germination and bioactivity in comparison with incubation at 20 or 25°C. CONCLUSION: This study indicates that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Formulations based on production of the high conidia yield may not result in optimal bioactivity and there is a trade-off between quantity and quality of T. atroviride LU132 conidia. Conidium production was shown to be a continuous process, and increased under a dark/light regime. This is the first report of bimodal conidium production in a Trichoderma biological control agent (BCA), which is likely to be on 20 days cycle, and is dependent on colony age rather than abiotic factors. Conidia produced after 15 days are likely to be the most suitable for use in commercial production of this strain as a BCA. SIGNIFICANCE AND IMPACT OF THE STUDY: Most studies on Trichoderma-based BCA have only shown the effect of culture conditions on the high conidia yield regardless of conidium quality. This study is the first report on conidium quality affected by principal culture conditions for Trichoderma biological control formulations.

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and ß-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.

[Cultivation of a novel cellulase/xylanase producer, Trichoderma longibrachiatum mutant TW 1-59-27: production of the enzyme preparation and the study of its properties].

As a result of gamma-mutagenesis of Trichoderma longibrachiatum TW1 and the subsequent selection of improved producers, a novel mutant strain, TW1-59-27, capable of efficiently secreting cellulase and xylanase was obtained. In a fed-batch cultivation, the new TW1-59-27 mutant was significantly more active compared with the original TW1 strain. For instance, the activities of cellulase (towards carboxymethylcellulose) and xylanase in the culture broth (CB) increased by 1.8 and two times, respectively, and the protein content increased by 1.47 times. The activity of these enzymes in the dry enzyme preparation derived from the CB of the TW1-59-27 mutant was 1.3-1.8 times higher than that in the preparation derived from the original TW1 strain. It was established that the cellulase from the enzyme preparation of the mutant strain demonstrated the maximum activity at 55-65 degrees C; it occurred in xylanase at 60 degrees C. The pH optima of these enzymes were pH 4.5-5.0 and pH 5.0-6.0, respectively. It was shown that the content of endoglucanases in the enzyme preparation increased from 7% to 13.5%; the effect is largely driven by the secretion of endoglucanase-1. An enzyme preparation with increased endoglucanase-1 content is promising for use as a feed additive in agriculture.

Oxidative damage induced by heat stress could be relieved by nitric oxide in Trichoderma harzianum LTR-2.

Trichoderma harzianum is an important commercial biocontrol fungal agent. The temperature has been shown to be an important parameter and strain-specific to the mycelia growth of fungi, but less report makes the known of the mechanisms in T. harzianum. In our study, a 6-h treatment of heat increased the thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) concentration in mycelia to 212 and 230 % the level of the control, respectively. The exogenous NO donor sodium nitroprusside (150 µM) reduced the TBARS concentration to 53 % of that under heat stress (HS). At the same time, the NO-specific scavenger at 250 µM, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxyl-3-oxide, prevented the exogenous NO-relieved TBARS accumulation under HS. The increased NO concentration under HS was reduced 41 % by the NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester, but not the nitrate reductase (NR) inhibitor tungstate. Our study exhibited that NO can protect the mycelia of T. harzianum from HS and reduce the oxidative damage by enhancing the activity of NOS and NR.

Efficiency of treatments for controlling Trichoderma spp during spawning in cultivation of lignicolous mushrooms

Trichoderma spp is the cause of the green mold disease in mushroom cultivation production. Many disinfection treatments are commonly applied to lignocellulose substrates to prevent contamination. Mushroom growers are usually worried about the contaminations that may occur after these treatments during handling or spawning. The aim of this paper is to estimate the growth of the green mold Trichoderma sp on lignocellulose substrates after different disinfection treatments to know which of them is more effective to avoid contamination during spawning phase. Three different treatments were assayed: sterilization (121 ºC), immersion in hot water (60 and 80 ºC), and immersion in alkalinized water. Wheat straw, wheat seeds and Eucalyptus or Populus sawdust were used separately as substrates. After the disinfection treatments, bagged substrates were sprayed with 3 mL of suspension of conidia of Trichoderma sp (10(5) conidia/mL) and then separately spawned with Pleurotus ostreatus or Gymnopilus pampeanus. The growth of Trichoderma sp was evaluated based on a qualitative scale. Trichoderma sp could not grow on non-sterilized substrates. Immersions in hot water treatments and immersion in alkalinized water were also unfavorable treatments for its growth. Co- cultivation with mushrooms favored Trichoderma sp growth. Mushroom cultivation disinfection treatments of lignocellulose substrates influence on the growth of Trichoderma sp when contaminations occur during spawning phase. The immersion in hot water at 60 ºC for 30 min or in alkalinized water for 36 h, are treatments which better reduced the contaminations with Trichoderma sp during spawning phase for the cultivation of lignicolous species.

Light-mediated control of gene expression in filamentous fungus Trichoderma reesei.

We developed a light-mediated system based on synthetic light-switchable transactivators. The transactivators bind promoter upon blue-light exposure and rapidly initiate transcription of target transgenes in filamentous fungus Trichoderma reesei. Light is inexpensive to apply, easily delivered, and instantly removed, and thus has significant advantages over chemical inducers.

Efficiency of treatments for controlling Trichoderma spp during spawning in cultivation of lignicolous mushrooms.

Trichoderma spp is the cause of the green mold disease in mushroom cultivation production. Many disinfection treatments are commonly applied to lignocellulose substrates to prevent contamination. Mushroom growers are usually worried about the contaminations that may occur after these treatments during handling or spawning. The aim of this paper is to estimate the growth of the green mold Trichoderma sp on lignocellulose substrates after different disinfection treatments to know which of them is more effective to avoid contamination during spawning phase. Three different treatments were assayed: sterilization (121 °C), immersion in hot water (60 and 80 °C), and immersion in alkalinized water. Wheat straw, wheat seeds and Eucalyptus or Populus sawdust were used separately as substrates. After the disinfection treatments, bagged substrates were sprayed with 3 mL of suspension of conidia of Trichoderma sp (10(5) conidia/mL) and then separately spawned with Pleurotus ostreatus or Gymnopilus pampeanus. The growth of Trichoderma sp was evaluated based on a qualitative scale. Trichoderma sp could not grow on non-sterilized substrates. Immersions in hot water treatments and immersion in alkalinized water were also unfavorable treatments for its growth. Co- cultivation with mushrooms favored Trichoderma sp growth. Mushroom cultivation disinfection treatments of lignocellulose substrates influence on the growth of Trichoderma sp when contaminations occur during spawning phase. The immersion in hot water at 60 °C for 30 min or in alkalinized water for 36 h, are treatments which better reduced the contaminations with Trichoderma sp during spawning phase for the cultivation of lignicolous species.

Targets of light signalling in Trichoderma reesei.

BACKGROUND: The tropical ascomycete Trichoderma reesei (Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. RESULTS: Our transcriptome analysis of strains lacking the photoreceptors BLR1 and BLR2 as well as ENV1 revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. We show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. In contrast to N. crassa, photoreceptor mediated regulation of carbon metabolism in T. reesei occurs primarily by BLR1 and BLR2 via their positive effect on induction of env1 transcription, rather than by a presumed negative effect of ENV1 on the function of the BLR complex. Nevertheless, genes consistently regulated by photoreceptors in N. crassa and T. reesei are significantly enriched in carbon metabolic functions. Hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved.Analysis of growth on different carbon sources revealed that the oxidoreductive D-galactose and pentose catabolism is influenced by light and ENV1. Transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by ENV1, BLR1 and/or BLR2. Additionally we detected an ENV1-regulated genomic cluster of 9 genes including the D-mannitol dehydrogenase gene lxr1, with two genes of this cluster showing consistent regulation in N. crassa. CONCLUSIONS: We show that one major output pathway of light signalling in Trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. Targets of ENV1 and BLR1/BLR2 are for the most part distinct and indicate individual functions for ENV1 and the BLR complex besides their postulated regulatory interrelationship.

The Trichoderma atroviride photolyase-encoding gene is transcriptionally regulated by non-canonical light response elements.

The BLR-1 and BLR-2 proteins of Trichoderma atroviride are the Neurospora crassa homologs of white collar-1 and -2, two transcription factors involved in the regulation of genes by blue light. BLR-1 and BLR-2 are essential for photoinduction of phr-1, a photolyase-encoding gene whose promoter exhibits sequences similar to well-characterized light regulatory elements of Neurospora, including the albino proximal element and the light response element (LRE). However, despite the fact that this gene has been extensively used as a blue light induction marker in Trichoderma, the function of these putative regulatory elements has not been proved. The described LRE core in N. crassa comprises two close but variably spaced GATA boxes to which a WC-1/-2 complex binds transiently upon application of a light stimulus. Using 5' serial deletions of the phr-1 promoter, as well as point mutations of putative LREs, we were able to delimit an ~ 50 bp long region mediating the transcriptional response to blue light. The identified light-responsive region contained five CGATB motifs, three of them displaying opposite polarity to canonical WCC binding sites. Chromatin immunoprecipitation experiments showed that the BLR-2 protein binds along the phr-1 promoter in darkness, whereas the application of a blue light pulse results in decreased BLR-2 binding to the promoter. Our results suggest that BLR-2 and probably BLR-1 are located on the phr-1 promoter in darkness ready to perform their function as transcriptional complex in response to light.

ENVOY is a major determinant in regulation of sexual development in Hypocrea jecorina (Trichoderma reesei).

Light is one crucial environmental signal which can determine whether a fungus reproduces asexually or initiates sexual development. Mating in the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) occurs preferentially in light. We therefore investigated the relevance of the light response machinery for sexual development in H. jecorina. We found that the photoreceptors BLR1 and BLR2 and the light-regulatory protein ENV1 have no effect on male fertility, while ENV1 is essential for female fertility. BLR1 and BLR2 were found to impact fruiting body formation although they are not essential for mating. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that BLR1, BLR2, and ENV1 negatively regulate transcript levels of both pheromone receptors as well as peptide pheromone precursors in light but not in darkness and in a mating type-dependent manner. The effect of BLR1 and BLR2 on regulation of pheromone precursor and receptor genes is less severe than that of ENV1 as strains lacking env1 show 100-fold (for ppg1) to more than 100,000-fold (for hpp1) increased transcript levels of pheromone precursor genes as well as more than 20-fold increased levels of hpr1, the pheromone receptor receiving the HPP1 signal in a MAT1-1 strain. ENV1 likely integrates additional signals besides light, and our results indicate that its function is partially mediated via regulation of mat1-2-1. We conclude that ENV1 is essential for balancing the levels of genes regulated in a mating-type-dependent manner, which contributes to determination of sexual identity and fruiting body formation.

Proteomic analysis of Trichoderma atroviride reveals independent roles for transcription factors BLR-1 and BLR-2 in light and darkness.

The genus Trichoderma is one of the most widely used biological control agents of plant-pathogenic fungi. The main mechanism for survival and dispersal of Trichoderma is through the production of asexual spores (conidia). The transition from filamentous growth to conidiation can be triggered by light, nutrient deprivation, and mechanical damage of the mycelium. We conducted proteomic profiling analyses of Trichoderma atroviride after a blue light pulse. The use of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis allowed us to identify 72 proteins whose expression was affected by blue light. Functional category analysis showed that the various proteins are involved in metabolism, cell rescue, and protein synthesis. We determined the relationship between mRNA levels of selected genes 30 min after a light pulse and protein expression levels at different times after the pulse and found this correlation to be very weak. The correlation was highest when protein and mRNA levels were compared for the same time point. The transcription factors BLR-1 and BLR-2 are vital to the photoconidiation process; here we demonstrate that both BLR proteins are active in darkness and affect several elements at both the transcript and protein levels. Unexpectedly, in darkness, downregulation of proteins prevailed in the Δblr-1 mutant, while upregulation of proteins predominated in the Δblr-2 mutant. Our data demonstrate that the BLR proteins play roles individually and as a complex.