Interfacial Self-Assembly of a Fungal Hydrophobin into a Hydrophobic Rodlet Layer

The Sc3p hydrophobin of the basidiomycete Schizophyllum commune is a small hydrophobic protein (100 to 101 amino acids) containing eight cysteine residues. Large amounts of the protein are excreted into the culture medium as monomers, but in the walls of aerial hyphae, the protein is present as an SDS-insoluble complex. In this study, we show that the Sc3p hydrophobin spontaneously assembles into an SDS-insoluble protein membrane on the surface of gas bubbles or when dried down on a hydrophilic surface. Electron microscopy of the assembled hydrophobin shows a surface consisting of rodlets spaced 10 nm apart, which is similar to those rodlets seen on the surface of aerial hyphae. When the purified Sc3p hydrophobin assembles on a hydrophilic surface, a surface is exposed with high hydrophobicity, similar to that of aerial hyphae. The rodlet layer, assembled in vivo and in vitro, can be disassembled by dissolution in trifluoroacetic acid and, after removal of the acid, reassembled into a rodlet layer. We propose, therefore, that the hydrophobic rodlet layer on aerial hyphae arises by interfacial self-assembly of Sc3p hydrophobin monomers, involving noncovalent interactions only. Submerged hyphae merely excrete monomers because these hyphae are not exposed to a water-air interface. The generally observed rodlet layers on fungal spores may arise in a similar way.     

Localization of Cladosporium fulvum hydrophobins reveals a role for HCf-6 in adhesion

Hydrophobins are amphipathic molecules which form part of fungal cell walls and extracellular matrices and perform a variety of roles in fungal growth and development. The tomato pathogen Cladosporium fulvum has six hydrophobin genes, HCf-1 to -6. We have devised an epitope tagging approach for establishing hydrophobin localization during growth in culture and in plants. In this paper we localize HCf-2, -3, -4 and -5 and compare the data to our previous observations for HCf-1 and -6. In culture, HCf-1, -2, -3 and 4 localize to conidia and also appear on aerial hyphae. HCf-4 is unique in that it appears on submerged hyphae. HCf-5 expression is tightly regulated and appears on aerial hyphae early on during growth. Only HCf-1, -3 and -6 were observed during infection; HCf-3 appears on both conidia and emerging germ tubes. We also show that HCf-6 is secreted and coats surfaces under and around growing hyphae and demonstrate the effect of deleting HCf-6 on the adhesion of germinating C. fulvum conidia to glass slides.     

Expression and ��-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP)

Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble β-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.     

A peroxidase gene family and gene trees in Heterobasidion and related genera

Four putative peroxidase-encoding gene fragments, named mnp1a, mnp1b, mnp2 and mnp3, were amplified with degenerative primers from the white-rot basidiomycete genus Heterobasidion. The fragments were cloned and sequenced. Similar fragments were produced and analyzed from the related genera Amylostereum, Bondarzewia and Echinodontium. Each amplified fragment contains three identically positioned introns. According to the predicted amino acid sequence, these fragments are most similar to two Mn peroxidase-encoding genes (MPGI and mnp2) and gene pgv of Trametes versicolor. Conserved residues thought to be essential for peroxidase function were identified. All four MnP gene loci of Heterobasidion were detected only in H. parviporum. Variation occurred in the predicted amino-acid sequences (131-132 amino acids) of all four fragments originating from the 47 Heterobasidion isolates tested. Amino acid variation in fragments of mnp2 and mnp3 separated European Heterobasidion parviporum ("S-type") and H. abietinum ("F-type"), known to have identical rDNA sequences. Asian and western North American isolates from fir, spruce and other hosts had the peroxidase amino acid sequences of European H. parviporum. American and European H. annosum ("P-type") isolates had different amino acid sequences and might be cryptic species.     

Four conserved intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis

Hydrophobins are morphogenetic proteins produced by fungi during assembly of aerial hyphae, sporulation, mushroom development and pathogenesis. Eight cysteine residues are present in hydrophobins and form intramolecular disulphide bonds. Here, we show that expressing eight cysteine-alanine substitution alleles of the MPG1 hydrophobin gene from Magnaporthe grisea causes severe defects in development of aerial hyphae and spores. Immunolocalization revealed that Mpg1 hydrophobin variants, lacking intact disulphide bonds, retain the capacity to self-assemble, but are not secreted to the cell surface. This provides the first genetic evidence that disulphide bridges in a hydrophobin are dispensable for aggregation, but essential for secretion.     

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.     

Hydrophobin Genes Involved in Formation of Aerial Hyphae and Fruit Bodies in Schizophyllum

Fungi typically grow by apical extension of hyphae that penetrate moist substrates. After establishing a branched feeding mycelium, the hyphae differentiate and grow away from the substrate into the air where they form various structures such as aerial hyphae and mushrooms. In the basidiomycete species Schizophyllum commune, we previously identified a family of homologous genes that code for small cysteine-rich hydrophobic proteins. We now report that the encoded hydrophobins are excreted in abundance into the culture medium by submerged feeding hyphae but form highly insoluble complexes in the walls of emerging hyphae. The Sc3 gene encodes a hydrophobin present in walls of aerial hyphae. The homologous Sc1 and Sc4 genes, which are regulated by the mating-type genes, encode hydrophobins present in walls of fruit body hyphae. The hydrophobins are probably instrumental in the emergence of these aerial structures.     

A novel putative partitivirus of the saprotrophic fungus Heterobasidion ecrustosum infects pathogenic species of the Heterobasidion annosum complex

We characterized the bisegmented genome of a putative double-stranded (ds) RNA virus from a Chinese isolate of the fungus Heterobasidion ecrustosum, a member of the Heterobasidion insulare species complex. The larger genomic segment of 1885bp encoded a putative RNA dependent RNA polymerase (RdRp, 585aa), and the smaller one for a putative coat protein of 521aa (1826bp). Phylogenetic analyses suggest that this novel virus species, named as 'Heterobasidion RNA virus 3 from H. ecrustosum, strain 1' (HetRV3-ec1), can be assigned to the family Partitiviridae, being most similar to the Helicobasidium mompa dsRNA mycovirus with RdRp amino acid similarity of 54%. The similarity to known viruses of other Heterobasidion species was notably low (25-39%). The virus could be experimentally transmitted to members of the Heterobasidion annosum complex: the European Heterobasidion abietinum and North American Heterobasidion occidentale, and the original host strain could be cured from the virus by thermal treatment. Microscopical observations showed that hyphae of H. ecrustosum anastomosed occasionally with H. abietinum and H. occidentale, and suggested a possible route for horizontal transmission between these sexually incompatible species. The virus infection seemed to cause variable effects on the growth rate of its fungal hosts, but the results were strongly dependent on fungal strain, growth medium and incubation temperature.     

The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene.

The spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent Mr = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.     

Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.     

Genetics and QTL mapping of somatic incompatibility and intraspecific interactions in the basidiomycete Heterobasidion annosum s.l

Somatic incompatibility (SI) is a system by which filamentous fungi can distinguish self from non-self by delimiting the own mycelia from that of other individuals of the same species. In this study, we show that SI in the basidiomycete Heterobasidion annosum sensu lato is controlled by four loci by observing the frequency of somatically compatible pairings in two experiments where isolates were paired in all possible combination. The first experiment utilized 63 heterokaryons each with one unique nucleus chosen from an array of sibling homokaryons paired with one unrelated nucleus of homokaryotic isolate TC-39-7. The second experiment used 39 heterokaryons each with one unique nucleus from the array of sibling homokaryons backcrossed with one of the parental strains (TC-122-12). We observed that SI allelic differences in a pairing alone are not enough to determine the degree of somatic incompatibility. In the first experiment, we also observed other interactions such as hyphal walls in interaction zones, increased exudation of dark-coloured metabolites and increased production of aerial hyphae. QTLs for the respective traits were positioned to a genetic linkage map of the H. annosum genome. Map-based cloning of the corresponding loci will shed much new light on intraspecific interactions in basidiomycetes.     

Heterologous array analysis in Heterobasidion: hybridisation of cDNA arrays with probe from mycelium of S, P or F-types

Because of the close relatedness between three species of Heterobasidion annosum (P-type), Heterobasidion parviporum (S-type) and Heterobasidion abietinum (F-type), we investigated the possible use of arrays from one species for studies of gene expression in the other. Clones containing partial cDNAs from 94 identifiable genes expressed during spore germination and differentiation in H. parviporum were printed manually in six replications on nylon membranes. The membrane was hybridized with chemifluorescent labelled cDNA from actively growing mycelia of H. parviporum, H. annosum or H. abietinum, cultivated on a non-selective substrate. Product-moment correlation coefficient varied between 0.81 and 0.49. Due to the level of correlation, in the gene expression among the intersterility groups, we concluded that the cDNA array of one can be used to study gene expression in the others.     

The properties of citrate transport catalyzed by CitP of Lactococcus lactis ssp. lactis biovar diacetylactis

Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.     

How a fungus escapes the water to grow into the air

Fungi are well known to the casual observer for producing water-repelling aerial moulds and elaborate fruiting bodies such as mushrooms and polypores. Filamentous fungi colonize moist substrates (such as wood) and have to breach the water-air interface to grow into the air. Animals and plants breach this interface by mechanical force. Here, we show that a filamentous fungus such as Schizophyllum commune first has to reduce the water surface tension before its hyphae can escape the aqueous phase to form aerial structures such as aerial hyphae or fruiting bodies. The large drop in surface tension (from 72 to 24 mJ m-2) results from self-assembly of a secreted hydrophobin (SC3) into a stable amphipathic protein film at the water-air interface. Other, but not all, surface-active molecules (that is, other class I hydrophobins and streptofactin from Streptomyces tendae) can substitute for SC3 in the medium. This demonstrates that hydrophobins not only have a function at the hyphal surface but also at the medium-air interface, which explains why fungi secrete large amounts of hydrophobin into their aqueous surroundings.     

SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating-type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a DeltaSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon-specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a DeltaSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.     

Identification, Characterization, and In Situ Detection of a Fruit-Body-Specific Hydrophobin of Pleurotus ostreatus

Hydrophobins are small (length, about 100 ± 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus. In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.     

The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.