Erzsébet Fekete

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: A master regulator of carbon assimilation

BackgroundThe identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known.ResultsHere, we deciphered the CRE1 regulatory range in the model cellulose and hemicellulose-degrading fungus Trichoderma reesei (anamorph of Hypocrea jecorina) by profiling transcription in a wild-type and a delta-cre1 mutant strain on glucose at constant growth rates known to repress and de-repress CCR-affected genes. Analysis of genome-wide microarrays reveals 2.8% of transcripts whose expression was regulated in at least one of the four experimental conditions: 47.3% of which were repressed by CRE1, whereas 29.0% were actually induced by CRE1, and 17.2% only affected by the growth rate but CRE1 independent. Among CRE1 repressed transcripts, genes encoding unknown proteins and transport proteins were overrepresented. In addition, we found CRE1-repression of nitrogenous substances uptake, components of chromatin remodeling and the transcriptional mediator complex, as well as developmental processes.ConclusionsOur study provides the first global insight into the molecular physiological response of a multicellular fungus to carbon catabolite regulation and identifies several not yet known targets in a growth-controlled environment.

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose.

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d‐galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d‐galactose, l‐arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d‐galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d‐galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta‐gal1 strain. In this pathway, galactitol is catabolised by the lad1‐encoded l‐arabinitol‐4‐dehydrogenase, because a gal1/lad1 double delta‐mutant failed to grow on d‐galactose. In the delta‐gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose‐1‐phosphate uridylyltransferase) by d‐galactose, but not by l‐arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose‐derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

Biodiversity and evolution of primary carbon metabolism in Aspergillus nidulans and other Aspergillus spp.

Some of the Aspergilli are reputed for their versatile and efficient catabolism of soluble carbon sources and related metabolites as well as raw polymeric materials. Here, we present a detailed investigation of the genomic and evolutionary basis for this versatility, using seven Aspergillus and one Neosartorya genome sequences. We manually annotated about 155 genes per genome covering glycolysis, the pentose phosphate cycle, alternative routes of D-glucose metabolism, catabolism of D-galactose and pentoses, and the TCA cycle, as well as the utilization of acetate and ethanol, propionate metabolism, and gluconeogenesis.The annotation reveals that the Aspergilli have re-enforced several areas of their primary metabolism(notably glycolysis, TCA cycle, ethanol utilization, and pentose and polyol metabolism) by gene duplications,horizontal gene transfer or gene clustering. Results from the phylogenetic analysis of several enzymes encoded by duplicated genes also suggests that some gene products may have acquired new(physiological) functions, that render primary carbon metabolism of the Aspergilli more complex than previously thought.

The VELVET A orthologue VEL1 of Trichoderma reesei regulates fungal development and is essential for cellulase gene expression.

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.

Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): A key to cellulase gene expression on lactose

The heterodisaccharide lactose (1,4-O-β-d-galactopyranosyl-d-glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei). Lactose assimilation is slow, and the assimilation of its β-d-galactose moiety depends mainly on the operation of a recently described reductive pathway and depends less on the Leloir pathway, which accepts only α-d-galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular β-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the β-anomer of d-galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.

Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.

Sexual Recombination in the Botrytis cinerea Populations in Hungarian Vineyards

Botrytis cinerea (anamorph of Botryotinia fuckeliana) causes gray mold on a high number of crop plants including grapes. In this study, we investigated the genetic properties of a grape pathogenic population of B. cinerea in the area of Eger, Hungary. A total of 109 isolates from 12 areas were sampled. Based on the sequence of the beta-tubulin (tub1) locus, they all belong to group II, a phylogenetic species within B. cinerea. Seventy-four isolates were classified as transposa, with both the Flipper and Boty transposons, and 10 were classified as vacuma, lacking both transposons. The remaining isolates contained either only Flipper (13) or Boty (12). Multilocus analysis of sequences from tub1 and two other loci (elongation factor 1-alpha, tef1, and a minisatellite from the intron of an ATPase, MSB1) led to poor phylogenetic resolution of strains in individual clades. Analysis of five microsatellites (Bc2, Bc3, Bc5, Bc6, and Bc10) resulted in 55 microsatellite haplotypes within the 109 strains. No correlation was detected among individual haplotypes and the presence/absence of Flipper and/or Boty, the geographic origin, or the year of isolation. Application of the index of association, the chi-square test, and the phi test consistently indicated that the population of Hungarian isolates of B. cinerea undergoes sexual reproduction. However, the index of association test suggested the presence of some clonality, and the fixation index showed a low or occasionally moderate level of fixation in the Flipper populations. We conclude that the B. cinerea populations in Hungary consist of a strongly recombining group II phylogenetic species.

Regulation of formation of the intracellular β-gaiactosidase activity ofAspergillus nidulans

The regulation of formation of the single intracellular β-galactosidase activity ofAspergillus nidulans was investigated. β-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose orD-galactose.L-Arabinose, and — with lower efficacy —D-xylose also induced β-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in β-galactosidase activity. In contrast, in cultures growing onD-galactose, addition of glucose decreased the activity of β-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not ofD-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of β-galactosidase and partially interfered with the induction of β-galactosidase byD-galactose,L-arabinose, andD-xylose.D-Galactose phosphorylation was not necessary for β-galactosidase induction, since induction byD-galactose occurred in anA. nidulans mutant defective in galactose kinase, and by the non-metabolizableD-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced β-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible byD-galactose, whereas it was still inducible byL-arabinose. We conclude that biosynthesis of the intracellular β-galactosidase ofA. nidulans} is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced byD-galactose andL-arabinose in independent ways.