Recent advances and future prospects in peptaibiotics, hydrophobin, and mycotoxin research, and their importance for chemotaxonomy of Trichoderma and Hypocrea

Fungi of the genus Trichoderma with teleomorphs in Hypocrea are abundant producers of a group of amphiphilic, non-ribosomal peptide antibiotics, which are rich in the non-proteinogenic amino acid Aib (alpha-aminoisobutyric acid). They are referred to as peptaibiotics, or peptaibols, if a 1,2-amino alcohol is present at the C-terminus. Trichoderma/Hypocrea, like other ascomycetous fungi, also produce hydrophobins, a class of small, cysteine-rich proteins. Advanced soft ionization mass spectrometric techniques such as LC-CID-MS, LC-ESI-MS(n), and IC-MALDI-TOF-MS enabled the high-throughput analysis, simultaneous detection and sequence determination of peptaibiotics and hydrophobins from minute quantities of fungal materials. Some Trichoderma species have been recognized to produce peptaibiotics as well as simple mycotoxins of the trichothecene group. The combination of sequence data of both groups of peptides with the pattern of low-molecular-weight secondary metabolites, including trichothecene-type mycotoxins, independently confirmed the results of morphological, molecular, and phylogenetic analyses. This approach established a new lineage in Trichoderma/Hypocrea, the Brevicompactum clade, comprising four new and one redescribed species. Notably, commercial preparations of single or mixed cultures of Trichoderma species, in particular T. harzianum, and T. koningii, are registered as biocontrol agents for soil and plant pathogens. In this context, it is emphasized that the four mycotoxin-producing species of the recently established Brevicompactum clade (T. brevicompactum, T. arundinaceum, T. turrialbense, and T. protrudens) are not closely related to any of the Trichoderma species currently used as biocontrol agents. Furthermore, possible health concerns about release of peptaibiotics in the biosphere are discussed with respect to their bioactivities and their use as drugs in human and veterinary medicine. Finally, future prospects regarding novel bioactivities and further research needs, including interdisciplinary taxonomic approaches, are outlined.     

Behavior of Trichoderma reesei hydrophobins in solution: interactions, dynamics, and multimer formation

Filamentous fungi utilize small amphiphilic proteins called hydrophobins in their adaptation to the environment. The hydrophobins are used to form coatings on various fungal structures, lower the surface tension of water, and to mediate surface attachment. Hydrophobins function through self-assembly at interfaces, for example, at the air-water interface, and at fungal cellular structures. Despite their high tendency to self assemble at interfaces, hydrophobins can be very soluble in water. To understand the mechanism of hydrophobin self-assembly, in this work, we have studied the behavior of two Trichoderma reesei hydrophobins, HFBI and HFBII in aqueous solution. The main methods used were F?rster resonance energy transfer (FRET) and size exclusion chromatography. A genetically engineered HFBI variant, NCys-HFBI, was utilized for the site-specific labeling of dyes for the FRET experiments. We observed the multimerization of HFBI in a concentration-dependent manner. A change from monomers to tetramers was seen when the hydrophobin concentration was increased. Interaction studies between HFBI and HFBII suggested that at low concentrations homodimers are preferred, and at higher concentrations, the heterotetramers of HFBI and HFBII are formed. In conclusion, the results support the model where hydrophobins in aqueous solutions form multimers by hydrophobic interactions. In contrast to micelles formed by detergents, the hydrophobin multimers are defined in size and involve specific protein-protein interactions.     

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma . To this end, we isolated 482 strains of Hypocrea / Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea / Trichoderma ( TrichO KEY), sequence similarity analysis ( Tricho blast ) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea / Trichoderma species from sections Trichoderma and Pachybasium , comprising H. lixii/T. harzianum , T. gamsii , T. spirale , T. velutinum , T. hamatum , H. koningii/T. koningii , H. virens/T. virens , T. tomentosum , H. semiorbis , H. viridescens/T. viridescens , H. atroviridis/T. atroviride , T. asperellum , H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum–Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum . All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea / Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.     

Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre‐purification by foam separation during cultivation

Hydrophobins are small surface‐active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor‐made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3‐phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha‐factor prepro‐peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre‐purification using foam separation.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering

Hydrophobins are amphiphilic proteins produced by filamentous fungi. They function in a variety of roles that involve interfacial interactions, as in growth through the air-water interface, adhesion to surfaces, and formation of coatings on various fungal structures. In this work, we have studied the formation of films of the class II hydrophobin HFBI from Trichoderma reesei at the air-water interface. Analysis of hydrophobin aqueous solution drops showed that a protein film is formed at the air-water interface. This elastic film was clearly visible, and it appeared to cause the drops to take unusual shapes. Because adhesion and formation of coatings are important biological functions for hydrophobins, a closer structural analysis of the film was made. The method involved picking up the surface film onto a solid substrate and imaging the surface by atomic force microscopy. High-resolution images were obtained showing both the hydrophilic and hydrophobic sides of the film at nanometer resolution. It was found that the hydrophobin film had a highly ordered structure. To study the orientation of molecules and to obtain further insight in film formation, we made variants of HFBI that could be site specifically conjugated. We then used the avidin-biotin interaction as a probe. On the basis of this work, we suggest that the unusual interfacial properties of this type of hydrophobins are due to specific molecular interactions which lead to an ordered network of proteins in the surface films that have a thickness of only one molecule. The interactions between the proteins in the network are likely to be responsible for the unusual surface elasticity of the hydrophobin film.     

Crystal structures of hydrophobin HFBII in the presence of detergent implicate the formation of fibrils and monolayer films

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2A resolution, R = 22, R(free) = 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 A resolution, R = 13, R(free) = 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.     

Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.     

Interaction and comparison of a class I hydrophobin from Schizophyllum commune and class II hydrophobins from Trichoderma reesei

Hydrophobins fulfill a wide spectrum of functions in fungal growth and development. These proteins self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. Hydrophobins are divided into two classes based on their hydropathy patterns and solubility. We show here that the properties of the class II hydrophobins HFBI and HFBII of Trichoderma reesei differ from those of the class I hydrophobin SC3 of Schizophyllum commune. In contrast to SC3, self-assembly of HFBI and HFBII at the water-air interface was neither accompanied by a change in secondary structure nor by a change in ultrastructure. Moreover, maximal lowering of the water surface tension was obtained instantly or took several minutes in the case of HFBII and HFBI, respectively. In contrast, it took several hours in the case of SC3. Oil emulsions prepared with HFBI and SC3 were more stable than those of HFBII, and HFBI and SC3 also interacted more strongly with the hydrophobic Teflon surface making it wettable. Yet, the HFBI coating did not resist treatment with hot detergent, while that of SC3 remained unaffected. Interaction of all the hydrophobins with Teflon was accompanied with a change in the circular dichroism spectra, indicating the formation of an alpha-helical structure. HFBI and HFBII did not affect self-assembly of the class I hydrophobin SC3 of S. commune and vice versa. However, precipitation of SC3 was reduced by the class II hydrophobins, indicating interaction between the assemblies of both classes of hydrophobins.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.     

Solution structure and interface‐driven self‐assembly of NC2, a new member of the Class II hydrophobin proteins

Hydrophobins are fungal proteins that self‐assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin‐based applications. Proteins 2014; 82:990–1003. © 2013 Wiley Periodicals, Inc.     

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1(-) gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1(-) mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1(-) mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin-encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.     

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract Eleven strains of Trichoderma viride , 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica , using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum , a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.     

Aggregation and self-assembly of hydrophobins from Trichoderma reesei: low-resolution structural models

Hydrophobins are secreted fungal proteins, which have diverse roles in fungal growth and development. They lower the surface tension of water, work as adhesive agents and coatings, and function through self-assembly. One of the characteristic properties of hydrophobins is their tendency to form fibrillar or rod-like aggregates at interfaces. Their structure is still poorly known. In a step to elucidate the structure/function relation of hydrophobin self-assembly, we present the low-resolution structure of self-assembled fibrils of the class II hydrophobin HFBII from Trichoderma reesei based on small and wide-angle x-ray scattering. We first studied the solution state (10 mg/mL) of both HFBI and HFBII and showed that they formed assemblages in aqueous solution, which have a radius of gyration of ~24 A and maximum dimension of ~65 A, corresponding to the size of a tetramer. This result was supported by size-exclusion chromatography. Undried samples of HFBII fibrils had a monoclinic crystalline structure, which changed to hexagonal when the material was dried. A low-resolution structure for the HFBII fibrils is suggested. There are data in the literature based on staining properties suggesting that hydrophobins of class I form assemblies with an amyloid structure. Comparison of the HFBII data (x-ray results, staining with thioflavin T) to published data showed that the HFBII assemblages are not amyloid.     

The hydrophobin HCf-1 of Cladosporium fulvum is required for efficient water-mediated dispersal of conidia

Six hydrophobin genes (HCf-1 to -6) have thus far been identified in the tomato pathogen Cladosporium fulvum. HCf-1 to -4 are Class I hydrophobins and HCf-5 and -6 are Class II hydrophobins. In this paper we describe the isolation of deletion mutants that lack HCf-1, HCf-2, or both these genes. Global down-regulation of the expression of Class I hydrophobins is achieved by homology-dependent gene silencing. Analysis of the mutant strains shows that HCf-1 confers hydrophilic character to the conidia and this facilitates the dissemination of conidia on the surface of water droplets. Other Class I hydrophobins, such as HCf-3 or HCf-4, may be involved in the development and germination of conidia.     

木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组、Trichoderma 组和Pachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma 组和Pachybasium组的所有菌株可分成两个群(A,B),B群进一步分为4个分支(B1,B2,B3,B4);A群由Trichoderma 组的H. aureoviridis和未鉴定到种的3个Hypocrea菌株构成;B1,B2,B4群均由Pachybasium组菌株构成;B3群由Pachybasium组的T. hamatum、T. strigosum和Trichoderma 组的T. asperellum、T. atroviride、T. koningii、T. viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma 组中的T. viride aggr.、T. atroviride、T. koningii归并入Pachybasium组,对Trichoderma 组重新定义。     

Differential Regulation and Posttranslational Processing of the Class II Hydrophobin Genes from the Biocontrol Fungus Hypocrea atroviridis

Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.Hydrophobins are unique and ubiquitous small proteins, characterized by the presence of eight positionally conserved cysteine residues, and present in all multicellular asco- and basidiomycetes. According to their hydropathy profiles and spacing between the conserved cysteines (37), they are divided into two classes (class I and class II). Hydrophobins are secreted proteins, found on the outer surfaces of the cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment (18, 24, 37), such as surface recognition during pathogenic interaction with plants, insects, or other fungi, but also in symbiosis (38). In addition, they also influence cell wall composition (33). Because of these manifold roles, it is less surprising that the expression of hydrophobin genes is subject to complex patterns of signals, including those that are related to the triggering of conidiogenesis or indicating the presence of a plant host.Many species of the fungal genus Hypocrea/Trichoderma are known as mycoparasites, and several of them are therefore applied as biocontrol agents (6, 7, 36). In addition, Trichoderma spp. have recently been reported to occur as endophytes and to be able to elicit positive plant responses against potential pathogens (17). Because of the reasons given above, hydrophobins would be candidate proteins playing a role in this process, and in fact a class I hydrophobin gene has recently been reported to be overproduced during endophytic interaction of Trichoderma asperellum and cucumber roots (35). In addition, other hydrophobins may be involved in the mechanism of mycoparasitism itself as well as the colonization of decaying wood.Our information about the roles of hydrophobins in the physiology of Trichoderma as well as other ascomycetous fungi is mostly derived from reversed genetics of a few major members (3, 4, 19-22). In Hypocrea jecorina (= Trichoderma reesei), two major class II hydrophobins (HFB-1 and HFB-2) have been studied in detail (4) and shown to be formed under different physiological conditions (29). However, the genome sequence of H. jecorina contains six class II hfb genes (27), and the roles of HFB-3, HFB-4, HFB-5, and HFB-6 are yet unknown. In the biocontrol fungus Hypocrea atroviridis (formerly called “Trichoderma harzianum”), only a single hydrophobin gene has been characterized so far (srh1 [28]) and shown to be expressed mainly under conditions of sporulation. Consequently, very little is known about hydrophobins and their regulation in Trichoderma.We have recently reported that two species of the Trichoderma/Hypocrea genus, Hypocrea virens and Hypocrea atroviridis, have an exceptional high number of class II hydrophobin genes (i.e., 11 and 10 phylogenetically different genes, respectively [22]). Therefore, the objective of this work was to investigate whether all of them are in fact expressed and, if so, under which conditions. We thereby put emphasis on vegetative growth, mycoparasitic interaction, and different triggers of sporulation and on learning whether the sporulation- and stress-regulating proteins BLR1 and BLR2 (10, 15) play a role in this process.In addition, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to detect the respective proteins and to learn their mode of processing. It has previously been shown that direct solvent extraction of mycelia and spores of Ascomycetes in the process of sample preparation provides a small set of protein peaks in the range of 5,000 to 10,000 Da representing the hydrophobin inventory (27). Structural studies of hydrophobins from H. jecorina (2, 20, 30, 31), Schizophyllum commune (13), and Agaricus bisporus (26) have shown expected signal peptide cleavage but also unusual processing patterns, including cleavage after Arg and Pro, as well as C-terminal modification.     

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.     

Hypocrea peltata: a mycological Dr Jekyll and Mr Hyde?

Hypocrea peltata (Pezizomycotina, Hypocreales, Hypocreaceae) is a common, widespread essentially subtropical species, with an uncharacteristically large stroma and asci containing four large and four small bicellular ascospores. Its only anamorph consists of indehiscent aleuriospores; it does not form a Trichoderma anamorph, which is typical of most Trichoderma/Hypocrea species. Hypocrea peltata grows very well at 37 C. The large stromata and failure to form a Trichoderma anamorph could lead one to doubt its generic placement. However sequences of the internal transcribed spacer region (ITS), 28S nuclear large subunit (LSU) of rDNA and RNA polymerase subunit II (rpb2) regions indicate that it represents a unique lineage within Trichoderma/Hypocrea. ITS and rbp2 sequences derived from cultures of H. peltata are identical to the "unidentified Hypocreaceae" reported in the literature as being isolated from lung of a patient with non-fatal pulmonary fibrosis.