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The Fungus Trichoderma Regulates Submerged Conidiation Using the Steroid Pregnenolone Hui Lin, Michael Travisano, and Romas J. Kazlauskas ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00376 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016
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The Fungus Trichoderma Regulates Submerged Conidiation Using the Steroid Pregnenolone Hui Lin,† Michael Travisano,†,‡ and Romas J. Kazlauskas*,†, § †
The Biotechnology Institute, ‡Department of Ecology, Evolution & Behavior, and §Department
of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108, USA
Abstract: In previous work, we evolved a population of Trichoderma citrinoviride in liquid cultures to speed up its asexual development cycle. The evolved population, called T-6, formed conidia 3 times sooner and in >1000-fold greater numbers. Here we identify the steroid pregnenolone as a molecular signal for this different behavior. Media in which the ancestral T. citrinoviride population was grown (called ancestral spent media) contained a submerged conidiation inhibitor. Growing the evolved population T-6 in ancestral spent media eliminated the abundant formation of conidia. This inhibition depended on the amount and age of the ancestral spent medium and the time that the ancestral spent medium was added to the T-6 culture. Fractionation of the ancestral spent medium identified a hydrophobic inhibiting compound with a molecular weight less than 2000 g/mol. A combination of GC-MS, ELISA and reaction with cholesterol oxidase identified it as pregnenolone. Addition of pregnenolone to cultures of T-6 inhibited submerged conidiation by inhibiting formation of conidiophores, while ten other analogous steroids did not. Pregnenolone also inhibited submerged conidiation of Fusarium graminearum PH-1, a plant pathogen that causes head blight in wheat and barley. This identification of steroids as signal molecules in fungi creates opportunities to disrupt this signaling to control fungal behavior.
All eukaryotes (animals, fungi, plants) contain steroids, which are hydrophobic organic molecules with a characteristic four-ring structure.1, 2 Steroids and their metabolites have two main functions. The largest fraction of the steroid molecules serve as components of cell membranes that alter membrane fluidity. Cholesterol serves this role in vertebrates, while ergosterol serves this role in fungi. These steroids make up ~20% of the mass of membrane lipids. Second,
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steroids serve as hormones – cell signaling molecules to turn gene transcription on and off.1 These steroids act at very low concentrations, typically in the range of ng/mL, are structure specific and can diffuse across cell membranes to act at distant sites. For example, 17β-estradiol regulates the female reproductive cycle in animals;3 birth control pills contain analogs of this estrogen. On solid media, the asexual life cycle of filamentous fungi starts with germination of asexual spores (conidia) followed by vegetative growth of mycelia (Figure 1). Environmental signals including exposure to air and light, combined with stresses like temperature changes and nutrient depletion, cause the mycelia to develop specialized reproductive structures called conidiophores, which produce conidia.4 Dispersal of conidia to a new environment restarts the life cycle. The natural isolate Trichoderma citrinoviride (teleomorph Hypocrea schweinitzii, ancestor) grows in this manner on agar plates and produces abundant green conidia after a few days. In liquid media, Trichoderma spp. fungi often form chlamydospores (thick-walled vegetative cell that function as spores) instead of conidia.5 Indeed, Trichoderma citrinoviride, when grown in rich liquid media, formed numerous chlamydospores after 5 days (Supplementary Figure S1). At 3 days, before formation of chlamydospores, Trichoderma citrinoviride also formed a few conidia (~104 per mL), a behavior called submerged conidiation.6 The formation of these submerged conidia is the focus of this work.
Figure 1. The asexual life cycle of ancestral and selected populations of Trichoderma citrinoviride. Conidia (green color, bottom photo) germinate and grow as hyphae (top left image). Later, these hyphae develop specialized multicellular reproductive structures called conidiophores (blue color, top right image), which form conidia at their tips. The time required for this cycle in liquid media differs for the ancestral and selected populations as does the appearance of the cultures (see text for details). The blue and green colors have been added to the photos for clarity.
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Asexual sporulation (conidiation) requires the expression of thousands of genes and intercellular communication to generate specialized cells and coordinate conidia production.7 Genes wetA, brlA and abaA form a regulatory pathway for conidiation in Aspergillus nidulans and Neurospora crassa.4 However, the signal molecules for this regulation are unknown. Most research is focused on characterization of morphology and physiology and optimization of induction. In a few cases, hydrophobic organic molecules inhibit or induce conidia development. 1Octen-3-ol inhibits germination of conidia in Penicillium paneum,8 while a diterpenoid (conidiogenone) induces conidiation in Penicillium cyclopium.9 Linoleic acid derivatives regulate sexual and asexual spore development in Aspergillus nidulans.10 While no steroids have been reported to regulate conidiation in fungi, steroids regulate sexual development or sporulation in protists. The water mold Achlya ambisexualis produces the steroid antheridiol, which regulates the sexual development.11 Cytokinins and steroids can initiate sporulation in the protist Dictyostelium sp..12 In previous work, experimental evolution created a population of Trichoderma citrinoviride with an accelerated life cycle. Daily transfer of an aliquot of liquid culture to fresh media selected for more efficient transfer. This selection yielded a population (called T-6) that formed abundant conidia (>106 conidia/mL) in submerged cultures within 24 h (Figure 1) (unpublished data). If grown for five days, T-6 also formed chlamydospores (Supplementary Figure S1), but this work focuses on the conidiation at 24 h. Others have observed submerged conidiation of Trichoderma spp.,6, 13 especially in shaking cultures, but the large numbers of conidia in T-6 are remarkable. These two closely related populations – ancestral and T-6 evolved – develop differently under the same conditions and thus offer an opportunity to discover the molecular signals that control this biological phenomenon. Here, we identify the steroid pregnenolone in media from ancestral populations and show that pregnenolone inhibits the development of conidiophores in T-6.
RESULTS AND DISCUSSION Ancestral T. citrinoviride secretes a conidiation inhibitor. After one day of growth in liquid culture (rich media), ancestral T. citrinoviride formed mycelial pellets (ø ~1 mm) suspended in clear liquid. After three days the mycelial pellets were larger, but the liquid remained clear. Microscopic examination revealed no conidia or conidiophores (Supplementary Figure S2A). In
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contrast, after one day of growth in liquid media, evolved population T-6 formed small mycelial pellets (ø
