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

['Feng Cai', 'The Key Laboratory Of Plant Immunity', 'Jiangsu Provincial Key Lab Of Solid Organic Waste Utilization', 'Nanjing Agricultural University', 'Nanjing', 'Fungal Genomics Laboratory', 'Fungig', 'Institute Of Chemical', 'Environmental', 'Bioscience Engineering']

Date: 2022-01 

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.

Hydrophobins (HFBs) are unique, non-toxic small fungal proteins with outstandingly high surface activity. They modulate surface properties by self-assembling at interfaces and have a multitude of applications for drug delivery, biosensors, and cosmetics. To explore the applied potential of HFBs, we investigated their functions in the naturally HFB-enriched Trichoderma spp. fungi (Ascomycota). We show that HFBs unexpectedly specifically accumulate inside aerial hyphae, where they associate with lipid-enriched organelles and putatively line up the vacuolar structures, which contribute to the structure and longevity of aerial mycelium. The maximum of HFB synthesis and secretion happens before the sporulation, providing a massive extracellular matrix suitable for the protective coating of rapidly produced spores. Furthermore, HFBs are involved in the water sensing mechanism of spores and thus are linked to the dormancy/germination switch. Our results reveal that HFBs have a broad range of intracellular functions and are essential for fungal development and fitness.

Funding: The research in China was supported by grants from the Fundamental Research Funds for the Central Universities (KYXK2020) to QS, and the National Natural Science Foundation of China ( http://www.nsfc.gov.cn ) 31801939 to FC. The research in Austria was supported by the Austrian Science Foundation ( www.FWF.ac.at ), P25613-B20, to ISD and the Vienna Science and Technology Fund ( www.WWTF.at ), LS13-048, to ISD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Cai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Most fungi have only a few HFB-encoding genes (JGI Mycocosm, June 2020), although the genomes of some extremophilic species (Wallemia ichthyophaga [ 16 ]) or mycorrhizal mushrooms [ 17 ] contain exceptionally rich arsenals of HFBs. In Ascomycota molds, the genomes of fungi from the order Hypocreales have an extreme variety of HFB-encoding genes [ 18 ]. Among them, the genus Trichoderma exhibits the highest number and diversity of HFBs, which were reported as the main genomic hallmarks of these fungi [ 18 , 19 ]. The number of HFBs in individual Trichoderma species can range from seven in T. reesei to 16 in T. atroviride [ 18 ]. Contrary to HFBs in mushrooms and some airborne Ascomycota such as species of Penicillium and Aspergillus, most Trichoderma HFBs do not form rodlets or amyloids and are soluble in organic solvents and detergents [ 7 , 20 , 21 ]. An ecological genetic study of the two Trichoderma species, T. harzianum and T. guizhouense, revealed that HFB4 on the spore surface can control the preferential dispersal mode and spore survival [ 1 ]. However, to date, the biological or ecological role of the enrichment of HFBs in Trichoderma genomes has not been understood [ 18 ]. In the present study, we investigated the localization, secretion mechanisms, and function of HFBs during the development of two commonly occurring cosmopolitan sibling Trichoderma species, T. harzianum and T. guizhouense [ 1 , 22 , 23 ]. The intracellular localization of HFBs was also investigated in the recombinant methylotrophic yeast Pichia pastoris, which has no HFB-encoding genes in its genome [ 24 ].

Since fungi frequently need to produce large amounts of spores in a short period of time [ 15 ] and spores require a HFB coat, they need to rapidly secrete large amounts of these proteins [ 1 ] that have to be synthesized by sporulating and most commonly aerial hyphae. In molds, aerial hyphae are ephemeral structures [ 5 ] that are vulnerable to the environmental stressors in an open air and quickly become aged because they are deprived of nutrient and water absorption. Thus, aerial hyphae are frequently exposed to drought, UV radiation, and mechanical damage by rain, wind, or animals, and they may also need to grow against gravity. The secretion of HFBs during the formation of sporangia and spores is unlikely, as conidiogenesis is an energy-consuming task [ 15 ] that leaves few resources for the synthesis of accessory proteins required in large amounts. Under such circumstances, the mechanisms by which HFBs can be abundantly secreted are not known.

One billion years of evolution of filamentous fungi [ 6 ] has resulted in molecular adaptations to the physicochemical challenges associated with their lifestyle. For example, higher filamentous fungi commonly secrete hydrophobins (HFBs), which are unique small (usually < 20 kDa) amphiphilic and highly surface-active proteins [ 7 – 9 ] that are characterized by the presence of eight cysteine (Cys) residues, four of which form two Cys—Cys pairs. HFBs are believed to be initially secreted in a soluble form and then can spontaneously localize at the hydrophilic/hydrophobic interface, where they assemble into amphipathic layers [ 10 ] of varying solubility [ 7 ]. These layers significantly decrease the interfacial tension, thus allowing the hyphae to breach the liquid surface and grow into the air by forming buoyant colonies. Fruiting bodies, aerial hyphae, and spores are also largely coated by HFBs to reduce wetting, provide resilience to environmental stresses [ 1 , 11 ], promote the adhesion of spores and hyphae to hydrophobic surfaces or interactions with symbiotic partners [ 4 ], and influence growth and development [ 12 – 14 ].

The hydrophobicity of the body surface is essential for the fungal lifestyle. Fungi feed by secreting digestive enzymes and subsequently absorbing dissolved small molecules from the surrounding substrate. This nutritional strategy requires a hydrophilic surface. However, nonmotile spores of higher fungi need to be hydrophobic because they are passively dispersed by air or water. Thus, for reproduction, fungi grow out of the substrate and form biofilms–aerial hyphae and hydrophobic sporogenic structures (e.g., fruiting bodies, sporangia, and conidiophores) and spores [ 1 , 2 ] catching a usually short moment of environmental conditions suitable for the dispersal. The hydrophobicity of the spore or hyphal cell wall also influences their postdispersal biotic and abiotic interactions such as adhesion to substrates and their symbiotic partnerships [ 3 , 4 ]. Therefore, the ability to adjust and modulate the hydrophobicity of the body surface is crucial for fungal lifestyle and autecology [ 1 , 5 ].

Results

Our previous ecological genetic investigation revealed that HFB4 and HFB10 are highly significant for the fitness of the two sibling Trichoderma species [1]. In particular, HFB4 contributes to the anemophilous dispersal of T. guizhouense and evolves under positive natural selection pressure in T. harzianum, where it is likely associated with the affinity to pluviophilous (rain droplets) spore dispersal. The genomes of T. harzianum CBS 226.95 (Th) and T. guizhouense NJAU 4742 (Tg) contain eleven and nine HFB-encoding genes, respectively [1]. All these proteins have the characteristic arrangement of eight Cys residues and possess signal peptides, which indicate their affinity to the conventional secretory pathway through endoplasmic reticulum (ER) and extracellular vesicle trafficking.

To address the function of these genes during different stages of the life cycle, we first tested their expression at (i) spore germination, (ii) in submerged trophic hyphae, (iii) in aerial hyphae before conidiation, and (iv) in aerial conidiating mycelium (Table 1). In both species, the highest transcription level of hfb genes was recorded during the life stages that included the formation of aerial hyphae. A principal component analysis (PCA) of the expression profile of hfb genes confirmed the strong involvement of hfb4 and hfb10 in the development of aerial mycelium and spores of Tg and Th and a minor role of hfb3 (only noticeable at the conidiophore formation stage), followed by hfb2 (S1 Fig). The remaining genes of both species were silent at these developmental stages and are probably required either for sexual reproduction or biotic interactions. Consequently, we then constructed a library of hfb-deficient, hfb-overexpressing and fluorescently labeled mutants for the above four genes in both species (Table 2). As fluorescent protein tags may potentially influence the properties of HFBs [25], we predicted the properties of the fusion proteins using in silico 3D modeling and molecular dynamics (MD) simulation analysis for their behavior in water (S2 Fig). Based on this analysis, we selected the two fluorescent proteins (mRFP and YFP) and designed most optimal fusion constructs that correspond to the exposed position of the HFB hydrophobic patches with the highest probability (S2 Fig). To verify that off-site genetic transformation did not cause phenotypic differences, each genotype was represented by multiple mutants (two to five) and characterized based on at least a pair. Furthermore, we produced reverse complemented mutants in which fluorescently tagged and untagged HFB-encoding genes were reintroduced to the corresponding deletion mutants and compared their properties. The phenotypes of the mutants are shown in the S3 Fig. Briefly and as expected, the deletion of either hfb4 or hfb10 or both genes resulted in reduced conidiation and a “wetted hyphae” phenotype (impaired surface hydrophobicity) in both species [1]. The deletion of hfb2 did not result in phenotypic alterations.

Hydrophobins massively accumulate inside aerial hyphae In vivo epifluorescence microscopy of mutant strains expressing fluorescently labeled HFBs (fused with mRFP and YFP in different combinations in the two species) unexpectedly revealed that the formation of aerial hyphae was accompanied by massive intracellular accumulation of HFB4 and HFB10, respectively, which were stored in different types of membrane-bound vesicles and in a periplasmic or cell wall location (Fig 1). Additionally, HFBs were not visible in the cytoplasm and, also contrary to expectations, HFB4 and HFB10 were not associated with the cell wall. Even though both proteins were also detected extracellularly, HFB4 had a higher affinity for intracellular accumulation in stalk cells of conidiophores and phialides (sporogenic cells on conidiophores) (Figs 1 and S4) than HFB10. In contrast, HFB10 had a higher affinity than HFB4 to solid/liquid interfaces on the microscopy glass slide outside the cells (Fig 1). Another protein that was possible to visualize was HFB3 that also showed intracellular accumulation and was also visible on the surface of phialides specifically at collarettes (S5 Fig). As hfb3 had very low expression levels (Table 1) and consequently HFB3::mRFP was hardly visible in vivo, this protein did not contribute to the subsequent study. PPT PowerPoint slide

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TIFF original image Download: Fig 1. In vivo visualization of intracellular accumulation of fluorescently labeled hydrophobins in aerial hyphae and conidiophores of Trichoderma spp. (A) Aerial hyphae of T. harzianum Th hfb4::yfp- Th hfb10::mrfp accumulating HFB4::YFP and HFB10::mRFP in putative conventional and tubular vacuoles (dashed area) and the periplasm (arrows). (B) A 3D reconstruction of fluorescent overlay images of the mature conidiophore of T. guizhouense producing HFB4::mRFP and HFB10::YFP. The white light image of the same conidiophore is shown in an inset. https://doi.org/10.1371/journal.pgen.1009924.g001 The selective affinity of HFB4 and HFB10 to conidiophores (S4 Fig) suggested that their intracellular localization was linked to their function rather than caused by the potential “utilization” of an alien fusion construct in vacuoles. Nevertheless, to test the role of the fluorescent tag, we expressed mRFP (without HFBs) using the promoter and signal peptide of Tg HFB4 ( Tg P hfb4 ::mrfp). The resulting mRFP protein was secreted into the medium and was not retained in hyphae (S6 Fig). Thus, the intracellular localization of fusion proteins is not associated with the fluorescent tag. To test whether this behavior of HFB peptides would occur in other fungi, we overexpressed Trichoderma HFB-encoding genes in Komagataella pastoris (Saccharomycetales, syn. Pichia pastoris), which has no hfb genes in its genome [24]. The resulting mutants are listed in Table 2. Despite the presence of the signal prepropeptide of the α-mating factor from Saccharomyces cerevisiae, P. pastoris cells also massively accumulated HFBs intracellularly (S7 Fig). Together, these results indicate that the intracellular localization of fluorescently tagged HFBs in vesicle-like organelles and in the periplasm is linked to the properties of HFBs and is not led by the signal peptide or a tag.

Secretion of the massive extracellular HFB-enriched matrix precedes the formation of spore rings The massive accumulation of HFBs in Trichoderma colonies turning to the conidiation is visible without magnification if fluorescently labeled mutants are placed in a fluorescent imaging system, such as ChemiDoc MP (Bio-Rad, USA) (Fig 5A). The time course observation showed the coordinated appearance of HFB4 and HFB10 prior to conidiation along with the formation of conidiophores (S17 Fig). The complementary monitoring of hfb gene expression during conidiogenesis demonstrated that in both species at least four hfb genes (hfb2, hfb3, hfb4, and hfb10) were significantly (up to several thousand-fold in Th, S18 Fig) upregulated precisely at the beginning of conidiophore formation compared to the vegetative hyphal growth (Fig 5D). In both species, the expression of hfb3 was clearly apparent at this stage, while it was not considered significant when the entire aerial mycelium was tested (Table 1). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Colony architecture and dynamic HFB accumulation in aerial hyphae before and during conidiogenesis. (A) Fluorescent imaging of early conidiating T. guizhouense colony (48 hours) with mRFP- and YFP-labeled HFB4 and HFB10, respectively. The time course images of Tg and Th observed in ChemiDoc are shown in S17 Fig. (B) A 3D reconstruction of the same colony under epifluorescent microscope. Arrows point to the conidiophores with spore clumps. S3 Video shows detailed 3D animations of the extracellular matrices for both species. (C) A colony of T. guizhouense forming the sporulating ring after 96 hours of incubation. (D) Relative expression of hfbs during the fine time course of conidiogenesis (stages 1 to 6 correspond to the hyphal growth without conidia as 1, early compact pustula formation as 2, appearance of the conidial ring as 3 and 4, the vegetative growth beyond the conidial ring as 5, and dispersed conidiogenesis as 6, also see S18 Fig the boxed areas in C) of Tg quantified by qPCR. The values are normalized to those of the housekeeping gene tef1 and calculated in relation to aerial hyphae prior to conidiogenesis (stage 1). Horizontal bars indicate standard deviations. Green dots and white arrows right to the bars indicate spores and conidiophores appearing stages, respectively. Complementary results for Th are provided in S18 Fig. https://doi.org/10.1371/journal.pgen.1009924.g005 Observation of the intact colony surface (no added water) under epifluorescent microscope revealed an HFB-enriched protein matrix surrounding the spores and conidiophores (Fig 5A). A 3D reconstruction of the colony surface showed that the matrix has an uneven distribution of HFB4 and HFB10 (see the S3 Video), with HFB4 being more associated with the spores and HFB10 with the hyphae. Thus, the massive secretion of HFBs by aerial hyphae is likely a prerequisite for conidiation not only because they are essential spore protectants [1, 10] but also because intracellular HFBs aid the formation of all hydrophobic structures of fungal colonies, such as aerial hyphae, conidiophores, and spores.

[END]

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