Efecto de la combinación de Metarhizium anisopliae y Gliocladium virens sobre la oviposición, eclosión y emergencia de Aedes aegypti / Effect of the combination of Metarhizium anisopliae and Gliocladium virens on the oviposition, hatching and emergency of Aedes aegypti

Resumen: Objetivo: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. Material y métodos: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. Resultados: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. Conclusión: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.

Abstract: Objective: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. Materials and methods: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. Results: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. Conclusion: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.

[Effect of the combination of Metarhizium anisopliae and Gliocladium virens on the oviposition, hatching and emergency of Aedes aegypti]. / Efecto de la combinación de Metarhizium anisopliae y Gliocladium virens sobre la oviposición, eclosión y emergencia de Aedes aegypti.

OBJECTIVE: To evaluate the effect of the combination of Metarhizium anisopliae and Gliocladium virens, both with Aqua Reslin Super, on the oviposition, hatching and emergence of Aedes aegypti. MATERIALS AND METHODS: Evaluations were carried out to determine the effect of treatments impregnated on filter paper and exposed within plastic containers on the oviposition, hatching and emergency of Aedes aegypti. RESULTS: The results indicated that the fungus and insecticide combinations did not affect the oviposition behavior, but if the hatching of the eggs and the adult's emergency. CONCLUSIONS: With the results it can be concluded that the combination of fungi + insecticide can be a good option to be applied in oviposition sites with a view to the development of a lethal ovitrap.

OBJETIVO: Evaluar el efecto de la combinación de Metarhizium anisopliae y Gliocladium virens, ambos con Aqua Reslin Super, sobre oviposición, eclosión y emergencia de Aedes aegypti. MATERIAL Y MÉTODOS: Se realizaron evaluaciones para determinar el efecto de los tratamientos impregnados en papel filtro y expuestos dentro de recipientes de plástico sobre la oviposición, eclosión y emergencia de Aedes aegypti. RESULTADOS: Los resultados indicaron que las combinaciones hongo e insecticida no afectaron el comportamiento de oviposición, pero sí la eclosión de los huevos y la emergencia del adulto. CONCLUSIONES: Con los resultados se puede concluir que la combinación de hongos + insecticida puede ser una buena opción para aplicarse en sitios de oviposición con miras al desarrollo de una ovitrampa letal.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

[Bacterium Arthrobacter agilis UMCV2 and diverse amines inhibit in vitro growth of wood-decay fungi]. / La bacteria Arthrobacter agilis UMCV2 y diversas aminas inhiben el crecimiento in vitro de hongos destructores de madera.

The kingdom Fungi is represented by a large number of organisms, including pathogens that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin. The aim of our work was to characterize the antifungal activity in Arthrobacter agilis UMCV2 and diverse amines against wood-decaying fungi. Four fungal organisms (designated as UMTM) were isolated from decaying wood samples obtained from a forest in Cuanajo-Michoacán, México. Two of them showed a clear enzymatic activity of cellulases, xylanases and oxido-reducing enzymes and were identified as Hypocrea (UMTM3 isolate) and Fusarium (UMTM13 isolate). In vitro, the amines showed inhibitory effect against UMTM growth and one of the amines, dimethylhexadecylamine (DMA16), exhibited strong potential as wood preventive treatment, against the attack of decaying fungi.

A novel pullulanase from a fungus Hypocrea jecorina QM9414: production and biochemical characterization.

Pullulanase production from a fungus Hypocrea jecorina QM9414 that produces native extracellular hydrolases having industrial applications was carried out in a shaking flask culture containing 0.5% amylopectin at a pH of 6.50 at 300C. The enzyme was purified 11-fold by ammonium sulfate fractionation, anion-exchange and gel-filtration chromatographies with a yield of 10.12% and a specific activity of 1.36 +/- 0.14 U/mg protein. The molecular mass of pullulanase was estimated to be 130.56 kDa by PAGE and SDS-PAGE, indicating that the native enzyme was a monomer. The optimum pH and temperature for purified enzyme was 6.5 and between 35 degrees-65 degreesC, respectively. The Km values for amylopectin, starch and pullulan as substrates were 10.7, 15.5 and 38.4 mg/mL, respectively. The Vmax values were found to be 3.32, 3.32 and 3.82 deltaA/min for amylopectin, starch and pullulan, respectively. The enzyme was stable at 40-70 degreesC for 30 min, but lost about 33% of its activity at 80 degreesC and about 43% of activity at 90 degreesC and 100 degreesC for the same incubation period. Pullulanase activity was stimulated by CoC1(2), NiC1(2), KI, NaC1, MgC1(2), and LiSO4. The enzyme was slightly inhibited by urea, CaC1(2) and beta3-mercaptoethanol. The enyzmatic characteristics, substrate specificity and the products of hydrolysis indicated that the enzyme was similar to those of type II pullulanases.

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

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.

Taxon-specific metagenomics of Trichoderma reveals a narrow community of opportunistic species that regulate each other's development.

In this paper, we report on the in situ diversity of the mycotrophic fungus Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya) revealed by a taxon-specific metagenomic approach. We designed a set of genus-specific internal transcribed spacer (ITS)1 and ITS2 rRNA primers and constructed a clone library containing 411 molecular operational taxonomic units (MOTUs). The overall species composition in the soil of the two distinct ecosystems in the Danube floodplain consisted of 15 known species and two potentially novel taxa. The latter taxa accounted for only 1.5 % of all MOTUs, suggesting that almost no hidden or uncultivable Hypocrea/Trichoderma species are present at least in these temperate forest soils. The species were unevenly distributed in vertical soil profiles although no universal factors controlling the distribution of all of them (chemical soil properties, vegetation type and affinity to rhizosphere) were revealed. In vitro experiments simulating infrageneric interactions between the pairs of species that were detected in the same soil horizon showed a broad spectrum of reactions from very strong competition over neutral coexistence to the pronounced synergism. Our data suggest that only a relatively small portion of Hypocrea/Trichoderma species is adapted to soil as a habitat and that the interaction between these species should be considered in a screening for Hypocrea/Trichoderma as an agent(s) of biological control of pests.

From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach.

BACKGROUND: Hypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene. RESULTS: We used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions. CONCLUSIONS: We identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.

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.

Molecular regulation of arabinan and L-arabinose metabolism in Hypocrea jecorina (Trichoderma reesei).

Hypocrea jecorina (anamorph: Trichoderma reesei) can grow on plant arabinans by the aid of secreted arabinan-degrading enzymes. This growth on arabinan and its degradation product L-arabinose requires the operation of the aldose reductase XYL1 and the L-arabinitol dehydrogenase LAD1. Growth on arabinan and L-arabinose is also severely affected in a strain deficient in the general cellulase and hemicellulase regulator XYR1, but this impairment can be overcome by constitutive expression of the xyl1 encoding the aldose reductase. An inspection of the genome of H. jecorina reveals four genes capable of degrading arabinan, i.e., the alpha-L-arabinofuranosidase encoding genes abf1, abf2, and abf3 and also bxl1, which encodes a beta-xylosidase with a separate alpha-L-arabinofuranosidase domain and activity but no endo-arabinanase. Transcriptional analysis reveals that in the parent strain QM9414 the expression of all of these genes is induced by L-arabinose and to a lesser extent by L-arabinitol and absent on D-glucose. Induction by L-arabinitol, however, is strongly enhanced in a Deltalad1 strain lacking L-arabinitol dehydrogenase activity and severely impaired in an aldose reductase (Deltaxyl1) strain, suggesting a cross talk between L-arabinitol and the aldose reductase XYL1 in an alpha-L-arabinofuranosidase gene expression. Strains bearing a knockout in the cellulase regulator xyr1 do not show any induction of abf2 and bxl1, and this phenotype cannot be reverted by constitutive expression of xyl1. The loss of function of xyr1 has also a slight effect on the expression of abf1 and abf3. We conclude that the expression of the four alpha-L-arabinofuranosidases of H. jecorina for growth on arabinan requires an early pathway intermediate (L-arabinitol or L-arabinose), the first enzyme of the pathway XYL1, and in the case of abf2 and bxl1 also the function of the cellulase regulator XYR1.

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

BACKGROUND: The filamentous ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) 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. RESULTS: Analysis of the regulatory targets of the G-protein alpha subunit GNA1 in H. jecorina revealed a carbon source and light-dependent role in signal transduction. Deletion of gna1 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 Deltagna1 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. CONCLUSION: 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 Galpha subunit in appropriation of intracellular resources to environmental (especially nutritional) conditions.

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

BACKGROUND: 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 Hypocrea jecorina (anamorph Trichoderma reesei) little is known about the participants in this circuit. RESULTS: Analyses of proteins binding to the cellulase activating element (CAE) within the promotor of the cellobiohydrolase cbh2 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 Neurospora crassa and Met30p of Saccharomyces cerevisiae. Transcription of lim1 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, lim1 transcriptionally reacts to a switch from constant darkness to constant light and is subject to regulation by the light regulatory protein ENVOY. Thus lim1, 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 H. jecorina 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 H. jecorina revealed that the predominantly mycelial sulphate permease is lacking, thus supporting this hypothesis. CONCLUSION: 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.

Photostimulation of Hypocrea atroviridis growth occurs due to a cross-talk of carbon metabolism, blue light receptors and response to oxidative stress.

Light is a fundamental abiotic factor which stimulates growth and development of the majority of living organisms. In soil saprotrophic fungi, light is primarily known to influence morphogenesis, particularly sexual and asexual spore formation. Here we present a new function of light, the enhancement of mycelial growth. The photostimulated mycelial growth of the soil fungus Hypocrea atroviridis was detected on 17 (out of 95 tested carbon sources) carbohydrates and polyols, which are metabolically related to cellulose and hemicelluloses, and which are mainly available in the upper soil litter layer. This stimulation depends differently on the function of the two blue light receptor proteins BLR-1 and BLR-2, respectively, BLR-1 being responsible for carbon source selectivity and response to permanent light. Evocation of oxidative stress response in darkness imitates the photostimulation on nine of these carbon sources, and this effect was fully dependent on the function of BLR-1. We conclude that light in combination with the availability of litter-specific carbon sources serves as a signal for the fungus to be above ground, thereby stimulating fast growth in order to produce a maximum of propagules in the shortest time. We further deduce that this process involves oxidative stress response and the two blue light receptor proteins BLR-1 and BLR-2, the former playing the major role.

The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D-galactose catabolism and necessary for beta-galactosidase and cellulase induction by lactose.

The Hypocrea jecorina D-xylose reductase encoding gene xyl1 shows low basal transcript levels, and is induced by D-xylose, L-arabinose and L-arabinitol and, to a lesser extent, by lactose, D-galactose, galactitol and xylitol. The recombinantly expressed XYL1 catalyzes the NADPH-dependent reduction of the pentoses D-xylose and L-arabinose and the hexose D-galactose. Deletion of xyl1 slightly reduces growth on all carbon sources, but a significant decrease is found on D-xylose, L-arabinose and D-galactose. Similar to pentose degradation, XYL1 reduces D-galactose to galactitol in a recently identified second D-galactose pathway. Strains impaired in both D-galactose pathways are almost unable to grow on D-galactose. Deltaxyl1 strains show reduced growth on lactose and are impaired in beta-galactosidase expression and induction of the major cellobiohydrolase gene cbh1. A strain deleted in the cellulase regulator XYR1 is even more severely impaired in growth and beta-galactosidase expression on lactose, and does not produce any cbh1 transcript at all. In this strain, only a low basal level of xyl1 transcription is found on lactose. Galactitol, but not D-galactose is able to induce xyl1 transcription in a XYR1-independent manner. Our results show that the role of the H. jecorina XYL1 is not restricted to D-xylose catabolism and demonstrates its importance for induction of cellulases and beta-galactosidases.

Induction of extracellular beta-galactosidase (Bga1) formation by D-galactose in Hypocrea jecorina is mediated by galactitol.

The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 beta-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of beta-galactosidase at comparable growth rates. To know more about the actual inducer for beta-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.

Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina.

Xyr1 (xylanase regulator 1) of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) was recently demonstrated to play an essential role in the transcriptional regulation of the xyn1 (xylanase 1-encoding) gene expression. Consequently, this study reports on the deletion of the xyr1 gene from the H. jecorina genome. Comparative studies of the growth behavior of the different mutant strains (deleted and retransformed xyr1) grown on various carbon sources pointed to the strongly reduced ability of the xyr1 deletion strain to utilize D-xylose and xylan. Transcriptional analysis of the xyl1 (D-xylose reductase 1-encoding) gene as well as measurements of corresponding enzymatic activities gave evidence that Xyr1 takes part in the control of the fungal D-xylose pathway, in particular in the regulation of D-xylose reductase. It could be demonstrated that the uptake of D-xylose into the fungal cell is uninfluenced in the Deltaxyr1 strain. Furthermore, transcriptional regulation of the major hydrolytic enzyme-encoding genes xyn1 and xyn2 (xylanases 1 and 2), cbh1 and cbh2 (cellobiohydrolases 1 and 2), and egl1 (endoglucanase 1) is strictly dependent on Xyr1. Regulation of the respective genes via Xyr1 is not affected by the substances mediating induction (xylose, xylobiose, and sophorose) and is indispensable for all modes of gene expression (basal, derepressed, and induced). Moreover, Xyr1, it was revealed, activated transcriptional regulation of inducer-providing enzymes such as beta-xylosidase BXLI and beta-glucosidase BGLI but was not shown to be involved in the regulation of BGLII.

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.

D-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates.

Lactose (1,4-O-beta-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0.015 h(-1)) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0.015 h(-1) than d-galactose and induced cellulases even at growth rates up to 0.042 h(-1). Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0.030 h(-1). It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.

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.