The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins-hydrophobins (HFBs)-that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.

At least three families of hyphosphere small secreted cysteine-rich proteins can optimize surface properties to a moderately hydrophilic state suitable for fungal attachment.

The secretomes of filamentous fungi contain a diversity of small secreted cysteine-rich proteins (SSCPs) that have a variety of properties ranging from toxicity to surface activity. Some SSCPs are recognized by other organisms as indicators of fungal presence, but their function in fungi is not fully understood. We detected a new family of fungal surface-active SSCPs (saSSCPs), here named hyphosphere proteins (HFSs). An evolutionary analysis of the HFSs in Pezizomycotina revealed a unique pattern of eight single cysteine residues (C-CXXXC-C-C-C-C-C) and a long evolutionary history of multiple gene duplications and ancient interfungal lateral gene transfers, suggesting their functional significance for fungi with different lifestyles. Interestingly, recombinantly produced saSSCPs from three families (HFSs, hydrophobins and cerato-platanins) showed convergent surface-modulating activity on glass and on poly(ethylene-terephthalate), transforming their surfaces to a moderately hydrophilic state, which significantly favoured subsequent hyphal attachment. The addition of purified saSSCPs to the tomato rhizosphere had mixed effects on hyphal attachment to roots, while all tested saSSCPs had an adverse effect on plant growth in vitro. We propose that the exceptionally high diversity of saSSCPs in Trichoderma and other fungi evolved to efficiently condition various surfaces in the hyphosphere to a fungal-beneficial state.

Ecological Genomics and Evolution of Trichoderma reesei.

The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) is an efficient industrial cell factory for the production of cellulolytic enzymes used for biofuel and other applications. Therefore, researches addressing T. reesei are relatively advanced compared to other Trichoderma spp. because of the significant bulk of available knowledge, multiple genomic data, and gene manipulation techniques. However, the established role of T. reesei in industry has resulted in a frequently biased understanding of the biology of this fungus. Thus, the recent studies unexpectedly show that the superior cellulolytic activity of T. reesei and other Trichoderma species evolved due to multiple lateral gene transfer events, while the innate ability to parasitize other fungi (mycoparasitism) was maintained in the genus, including T. reesei. In this chapter, we will follow the concept of ecological genomics and describe the ecology, distribution, and evolution of T. reesei, as well as critically discuss several common misconceptions that originate from the success of this species in applied sciences and industry.

Molecular Identification of Trichoderma reesei.

Fungi comprise one of the most diverse groups of eukaryotes with many cryptic species that are difficult to identify. In this chapter, we detail a protocol for the molecular identification of the most industrially relevant species of Trichoderma-T. reesei. We first describe how a single spore culture should be isolated and used for the sequencing of the diagnostic fragment of the tef1 gene. Then, we provide two alternative methods that can be used for molecular identification and offer the diagnostic oligonucleotide hallmark of the tef1 sequence that is present in sequences of all T. reesei strains known to date and that is therefore suitable for reliable and straightforward identification.

Evolutionary compromises in fungal fitness: hydrophobins can hinder the adverse dispersal of conidiospores and challenge their survival.

Fungal evolutionary biology is impeded by the scarcity of fossils, irregular life cycles, immortality, and frequent asexual reproduction. Simple and diminutive bodies of fungi develop inside a substrate and have exceptional metabolic and ecological plasticity, which hinders species delimitation. However, the unique fungal traits can shed light on evolutionary forces that shape the environmental adaptations of these taxa. Higher filamentous fungi that disperse through aerial spores produce amphiphilic and highly surface-active proteins called hydrophobins (HFBs), which coat spores and mediate environmental interactions. We exploited a library of HFB-deficient mutants for two cryptic species of mycoparasitic and saprotrophic fungi from the genus Trichoderma (Hypocreales) and estimated fungal development, reproductive potential, and stress resistance. HFB4 and HFB10 were found to be relevant for Trichoderma fitness because they could impact the spore-mediated dispersal processes and control other fitness traits. An analysis in silico revealed purifying selection for all cases except for HFB4 from T. harzianum, which evolved under strong positive selection pressure. Interestingly, the deletion of the hfb4 gene in T. harzianum considerably increased its fitness-related traits. Conversely, the deletion of hfb4 in T. guizhouense led to the characteristic phenotypes associated with relatively low fitness. The net contribution of the hfb4 gene to fitness was found to result from evolutionary tradeoffs between individual traits. Our analysis of HFB-dependent fitness traits has provided an evolutionary snapshot of the selective pressures and speciation process in closely related fungal species.

The Evolutionary and Functional Paradox of Cerato-platanins in Fungi.

Cerato-platanins (CPs) form a family of fungal small secreted cysteine-rich proteins (SSCPs) and are of particular interest not only because of their surface activity but also their abundant secretion by fungi. We performed an evolutionary analysis of 283 CPs from 157 fungal genomes with the focus on the environmental opportunistic plant-beneficial and mycoparasitic fungus Trichoderma Our results revealed a long evolutionary history of CPs in Dikarya fungi that have undergone several events of lateral gene transfer and gene duplication. Three genes were maintained in the core genome of Trichoderma, while some species have up to four CP-encoding genes. All Trichoderma CPs evolve under stabilizing natural selection pressure. The functional genomic analysis of CPs in Trichoderma guizhouense and Trichoderma harzianum revealed that only epl1 is active at all stages of development but that it plays a minor role in interactions with other fungi and bacteria. The deletion of this gene results in increased colonization of tomato roots by Trichoderma spp. Similarly, biochemical tests of EPL1 heterologously produced by Pichia pastoris support the claims described above. Based on the results obtained, we conclude that the function of CPs is probably linked to their surfactant properties and the ability to modify the hyphosphere of submerged mycelia and, thus, facilitate the nutritional versatility of fungi. The effector-like functions do not sufficiently describe the diversity and evolution of these proteins in fungi, as they are also maintained, duplicated, or laterally transferred in the genomes of nonherbivore fungi.IMPORTANCE Cerato-platanins (CPs) are surface-active small proteins abundantly secreted by filamentous fungi. Consequently, immune systems of plants and other organisms recognize CPs and activate defense mechanisms. Some CPs are toxic to plants and act as virulence factors in plant-pathogenic fungi. Our analysis, however, demonstrates that the interactions with plants do not explain the origin and evolution of CPs in the fungal kingdom. We revealed a long evolutionary history of CPs with multiple cases of gene duplication and events of interfungal lateral gene transfers. In the mycoparasitic Trichoderma spp., CPs evolve under stabilizing natural selection and hamper the colonization of roots. We propose that the ability to modify the hydrophobicity of the fungal hyphosphere is a key to unlock the evolutionary and functional paradox of these proteins.

Stress Reshapes the Physiological Response of Halophile Fungi to Salinity.

(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillussydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.

Agricultural systems as potential sources of emerging human mycoses caused by Trichoderma: a successful, common phylotype of Trichoderma longibrachiatum in the frontline.

Trichoderma species are abundant in different agricultural habitats, but some representatives of this genus, mainly clade Longibrachiatum members are also emerging as causative agents of various human diseases with even fatal outcome. Strains of these species frequently show resistance to commonly used azole antifungals. Based on previous data it is hypothesized that Trichoderma isolates identified in human infections derive from environmental-including agricultural-origins. We examined Trichoderma longibrachiatum Rifai and Trichoderma bissettii Sandoval-Denis & Guarro strains recovered from four novel cases of human mycoses, along with isolates from previous case reports and different agricultural habitats, using multilocus phylogenetic analysis, BIOLOG Phenotype Microarrays and Etest. Strains attributed to T. bissettii were more abundant in both clinical and agricultural specimens compared to T. longibrachiatum. The majority of the isolates of both taxa could tolerate >256, >32 and >32 µg/ml fluconazole, itraconazole and posaconazole, respectively. None of the obtained results revealed characteristic differences between strains of clinical and agricultural origin, nor between the two taxa, supporting that agricultural environments may be significant sources of infections caused by these emerging human fungal pathogens. Furthermore, based on our findings we propose the re-classification of T. bissettii as T. longibrachiatum f. sp. bissettii.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Guttation capsules containing hydrogen peroxide: an evolutionarily conserved NADPH oxidase gains a role in wars between related fungi.

When resources are limited, the hypocrealean fungus Trichoderma guizhouense can overgrow another hypocrealean fungus Fusarium oxysporum, cause sporadic cell death and arrest growth. A transcriptomic analysis of this interaction shows that T. guizhouense undergoes a succession of metabolic stresses while F. oxysporum responded relatively neutrally but used the constitutive expression of several toxin-encoding genes as a protective strategy. Because of these toxins, T. guizhouense cannot approach it is potential host on the substrate surface and attacks F. oxysporum from above. The success of T. guizhouense is secured by the excessive production of hydrogen peroxide (H2 O2 ), which is stored in microscopic bag-like guttation droplets hanging on the contacting hyphae. The deletion of NADPH oxidase nox1 and its regulator, nor1 in T. guizhouense led to a substantial decrease in H2 O2 formation with concomitant loss of antagonistic activity. We envision the role of NOX proteins in the antagonism of T. guizhouense as an example of metabolic exaptation evolved in this fungus because the primary function of these ancient proteins was probably not linked to interfungal relationships. In support of this, F. oxysporum showed almost no transcriptional response to T. guizhouense Δnox1 strain indicating the role of NOX/H2 O2 in signalling and fungal communication.

Massive lateral transfer of genes encoding plant cell wall-degrading enzymes to the mycoparasitic fungus Trichoderma from its plant-associated hosts.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

Diverse Plant-Associated Pleosporalean Fungi from Saline Areas: Ecological Tolerance and Nitrogen-Status Dependent Effects on Plant Growth.

Similar to mycorrhizal mutualists, the rhizospheric and endophytic fungi are also considered to act as active regulators of host fitness (e.g., nutrition and stress tolerance). Despite considerable work in selected model systems, it is generally poorly understood how plant-associated fungi are structured in habitats with extreme conditions and to what extent they contribute to improved plant performance. Here, we investigate the community composition of root and seed-associated fungi from six halophytes growing in saline areas of China, and found that the pleosporalean taxa (Ascomycota) were most frequently isolated across samples. A total of twenty-seven representative isolates were selected for construction of the phylogeny based on the multi-locus data (partial 18S rDNA, 28S rDNA, and transcription elongation factor 1-α), which classified them into seven families, one clade potentially representing a novel lineage. Fungal isolates were subjected to growth response assays by imposing temperature, pH, ionic and osmotic conditions. The fungi had a wide pH tolerance, while most isolates showed a variable degree of sensitivity to increasing concentration of either salt or sorbitol. Subsequent plant-fungal co-culture assays indicated that most isolates had only neutral or even adverse effects on plant growth in the presence of inorganic nitrogen. Interestingly, when provided with organic nitrogen sources the majority of the isolates enhanced plant growth especially aboveground biomass. Most of the fungi preferred organic nitrogen over its inorganic counterpart, suggesting that these fungi can readily mineralize organic nitrogen into inorganic nitrogen. Microscopy revealed that several isolates can successfully colonize roots and form melanized hyphae and/or microsclerotia-like structures within cortical cells suggesting a phylogenetic assignment as dark septate endophytes. This work provides a better understanding of the symbiotic relationship between plants and pleosporalean fungi, and initial evidence for the use of this fungal group in benefiting plant production.

HFB7 - A novel orphan hydrophobin of the Harzianum and Virens clades of Trichoderma, is involved in response to biotic and abiotic stresses.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity.

Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture.

Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome.

Genome Sequence and Annotation of Trichoderma parareesei, the Ancestor of the Cellulase Producer Trichoderma reesei.

The filamentous fungus Trichoderma parareesei is the asexually reproducing ancestor of Trichoderma reesei, the holomorphic industrial producer of cellulase and hemicellulase. Here, we present the genome sequence of the T. parareesei type strain CBS 125925, which contains genes for 9,318 proteins.