bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.255406; this version posted August 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Intracellular accumulation and secretion of hydrophobin-enriched 2 vesicles aid the rapid sporulation of molds 3 Running title: Intracellular functions of hydrophobins in molds 4 Feng Cai1-3, Zheng Zhao2, Renwei Gao2, Mingyue Ding2, Siqi Jiang2, Qi Gao1, Komal Chenthamara3, 5 Marica Grujic3, Zhifei Fu4, Jian Zhang1, Agnes Przylucka3, Pingyong Xu 4, Günseli Bayram Akcapinar3,5, 6 Qirong Shen1*, Irina S. Druzhinina1-3* 7 1The Key Laboratory of Plant Immunity, Jiangsu Provincial Key Lab of Solid Organic Waste Utilization, 8 Nanjing Agricultural University, Nanjing, China 9 2Fungal Genomics Laboratory (FungiG), Nanjing Agricultural University, Nanjing, China 10 3Institute of Chemical, Environmental and Bioscience Engineering (ICEBE), TU Wien, Vienna, Austria 11 4Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Science, Beijing, China 12 5Department of Medical Biotechnology, Institute of Health Sciences, Acibadem Mehmet Ali Aydinlar 13 University, Istanbul, Turkey 14 *Corresponding authors: Irina S. Druzhinina [email protected], Qirong Shen 15 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.255406; this version posted August 18, 2020. The copyright holder for this preprint 16 Abstract(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 Fungi can rapidly produce large amounts of spores suitable for aerial dispersal. The hydrophobicity of 18 spores is provided by the unique amphiphilic and superior surface-active proteins – hydrophobins (HFBs) 19 – that self-assemble at hydrophobic/hydrophilic interfaces and thus change surface properties. Using the 20 HFB-enriched mold Trichoderma and the HFB-free yeast Pichia pastoris, we revealed a distinctive HFB 21 secretory pathway that includes an intracellular accumulation of HFBs in lipid bodies (LBs) that can 22 internalize in vacuoles. The resulting vacuolar multicisternal structures (VMS) are stabilized by HFB 23 layers that line up on their surfaces. These HFB-enriched VMSs can move to the periplasm for secretion 24 or become fused in large tonoplast-like organelles. The latter contributes to the maintenance of turgor 25 pressure required for the erection of sporogenic structures and rapid HFB secretion by squeezing out 26 periplasmic VMSs through the cell wall. Thus, HFBs are essential accessory proteins for the 27 development of aerial hyphae and colony architecture. 28 29 Key words: aerial hyphae, electron microscopy, extracellular vesicles, in vivo microscopy, intracellular 30 vesicles, lipid bodies, Pichia pastoris, small secreted cysteine-rich proteins, Trichoderma, vacuoles, 31 vacuolar multicisternal structures 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.255406; this version posted August 18, 2020. The copyright holder for this preprint 32 Introduction(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 33 The biology of filamentous fungi (mainly molds and mushrooms) is widely explained by the simple shape 34 of their body, which is an apically growing branching tube or hypha. The physiochemical properties of the 35 hyphal surface are important for fungal survival because fungi usually grow into their food or on the 36 surface and feed by secreting digestive enzymes and subsequently absorbing dissolved small molecules. 37 This nutritional strategy requires a large surface area/volume ratio (i.e., tubular shape) and a hydrophilic 38 surface. However, fungi reproduce by spores that are not motile and therefore passively dispersed by air 39 or water. Therefore, for reproduction and dispersal, fungi need to rapidly grow out of the substrate and 40 form aerial and hydrophobic sporogenic structures (e.g., fruiting bodies, sporangia, and conidiophores) 41 and spores1, 2 catching suitable dispersal conditions. The hydrophobicity of the spore or hyphal cell wall 42 also influences their adhesion to the substrate and their interactions with symbiotic partners3, 4. Thus, the 43 ability to modulate the hydrophobicity of the body surface and cross the hydrophilic/hydrophobic (i.e., 44 water/air) interface is crucial for fungal lifestyle5. 45 One billion years of evolution of filamentous fungi6 has resulted in molecular adaptations to the 46 physicochemical challenges associated with their lifestyle. For example, higher filamentous fungi 47 commonly secrete hydrophobins (HFBs), which are small (usually < 20 kDa) amphiphilic and highly 48 surface-active proteins7, 8, 9 that are characterized by the presence of eight cysteine (Cys) residues, and 49 four of these residues form two Cys - Cys pairs. HFBs are initially secreted in a soluble form and then 50 spontaneously localized at the hydrophilic/hydrophobic interface, where they assemble into amphipathic 51 layers10 of varying solubility7, 11. These layers significantly decrease the interfacial tension, thus allowing 52 the hyphae to breach the liquid surface and grow into the air. Fruiting bodies, aerial hyphae or spores 53 are also largely coated by HFBs to reduce wetting, provide resilience to environmental stresses2, 12, 54 promote the adhesion of spores and hyphae to hydrophobic surfaces or interactions with symbiotic 55 partners4, and influence growth and development 13, 14, 15. 56 Since fungi rapidly produce large amounts of spores16, they need to rapidly secrete large 57 amounts of HFBs that need to be synthesized by sporulating aerial hyphae. In molds, aerial hyphae are 58 ephemeral structures5, 17 that quickly become vulnerable and aged because they are deprived of nutrient 59 and water absorption. In addition, aerial hyphae are frequently exposed to drought, UV radiation, and 60 mechanical damage by rain, wind, or animals, and they may also need to grow against gravity. The 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.255406; this version posted August 18, 2020. The copyright holder for this preprint 61 massive(which formation was not certified of sporangia by peer review) and spores is the author/funder. is an energy-consuming All rights reserved. Notask reuse16 that allowed leaves without little permission. resource for 62 the secretion of HFBs. Under such circumstances, the mechanisms by which HFB can be massively 63 secreted and how this process coordinates with the rapid sporulation of the fungus, allowing these 64 proteins to play multiple roles in fungal reproduction, are not known. 65 Most fungi manage the above requirements with the aid of only a few HFB-encoding genes (JGI 66 Mycocosm, June 2020), although some extremophilic species (Wallemia ichthyophaga18) or mycorrhizal 67 mushrooms19 have a rich arsenal of HFBs. In Ascomycota molds, the genomes of fungi from the order 68 Hypocreales have an extreme variety of HFB-encoding genes20. Among them, the genus Trichoderma 69 exhibits the highest number and diversity of HFBs, which constitute the main genomic hallmarks of these 70 fungi20, 21. The number of HFBs in individual species can range from seven in T. reesei to 16 in T. 71 atroviride20. Compared with HFBs in mushrooms and some airborne Ascomycota, Trichoderma HFBs do 72 not form rodlets or amyloids and are soluble in organic solvents and detergents7, 11, 22. An ecological 73 genetic study of T. guizhouense and T. harzianum revealed that HFB4 on the spore surface controls the 74 preferential dispersal mode and spore survival2. However, to date, the biological role of the enrichment of 75 HFBs in the Trichoderma genomes has not been understood20. In the present study, we investigated the 76 localization secretion mechanisms and function of HFBs during the development of the two commonly 77 occurring cosmopolitan sibling Trichoderma species T. harzianum and T. guizhouense23, 24. The effect of 78 HFB production on the cell ultrastructure was also investigated in the methylotrophic yeast Pichia 79 pastoris, which has no HFB-encoding genes25. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.18.255406; this version posted August 18, 2020. The copyright holder for this preprint 80 Results(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 At least four HFBs are involved in the development of T. harzianum and T. guizhouense 82 The genomes of T. harzianum CBS 226.95 (Th) and T. guizhouense NJAU 4742 (Tg) contain eleven and 83 nine HFB-encoding genes, respectively2. An expression analysis performed at the four stages of the life 84 cycle (Supplementary material 1 Table S1.1) revealed that in both species, the highest transcription level 85 of hfb genes was recorded during the formation of aerial hyphae. A principal component analysis (PCA) 86 of the expression profile of hfb genes confirmed the strong involvement of hfb4 and hfb10 in the 87 development of Tg and Th and a minor role of hfb2 (Supplementary material 1 Figure S1.1). The 88 remaining genes were hardly expressed at all stages except hfb3, which was only noticeable at the 89 aerial hyphal formation stage. Thus, we constructed a library of hfb-deficient, hfb-overexpressing and 90 fluorescently labeled mutants for the above four genes (Supplementary material 2 Table S2.1). As 91 fluorescent protein