Vegetative growth and multicellular reproduction of Aspergillus nidulans

The growth of A. nidulans starts with the germination of an asexual conidiospore or a sexual ascospore, which is triggered by environmental signals. This leads to a network of vegetative hyphae, also called mycelium (Adams et al., 1998; Krijgsheld et al., 2011). The fungal spore generates an axis of polarization, before it forms a germ tube that develops to an elongated tubular structure (hypha). The formation of vegetative hyphae is the simplest form of growth, which allows fast colonization of new environments. Hyphal growth is promoted by apical extension (Harris et al., 2009; Virag et al., 2007). At a certain size, hyphae form septa to divide their cytoplasm into different compartments (Wolkow et al., 1996). Septae are formed by invagination of the plasma membrane and accumulation of cell wall material (Harris, 2001).

These so-called cross walls have pores, which allow the transfer of vesicles or nuclei between different compartments. In case of injury or stress, these pores can be closed by Woronin bodies to protect the not affected parts of the hyphae (Collinge and Markham, 1985; Timberlake, 1990).

The initial spore can develop more polarity axes and form secondary or tertiary germ tubes (Virag et al., 2007). In addition, hyphae are able to branch and form lateral tubular structures to build a close network (Harris, 2008). Different hyphal branches can fuse to each other to allow intercellular communication and nutrient exchange (Harris, 2008). This vegetative growth form continues in liquid media as long as enough nutrients are present (Krijgsheld et al., 2011).

A. nidulans reaches developmental competence after 16-20 hours (h) of growth (Axelrod et al., 1973). The fungus can then sense and react to environmental stimuli such as light, oxygen, temperature or pH and change gene expression and protein synthesis accordingly during this time (Axelrod et al., 1973; Bayram et al., 2016; Bayram and Braus, 2012).

A. nidulans enters in darkness and on an air-oxygen interface preferably the sexual life cycle, but develops also asexual conidiophores. Illumination induces the asexual conidiophore formation significantly, whereas the energy-consuming sexual life cycle is reduced (Bayram et al., 2016). The decision for the asexual or sexual life cycle is not only dependent on light, but also influenced by environmental signals such as CO2, O2, nutrients, pH or internal signals like pheromones (Axelrod et al., 1973; Bayram et al., 2016; Tsitsigiannis et al., 2004, 2005). The fungus enters the asexual life cycle during light exposure resulting in the formation of complex conidiophores, which produce the mitotically derived asexual conidiospores. Already 30 min exposure to light is sufficient to induce the asexual life cycle in fungal hyphae that reached the state of developmental competence (Adams et al., 1998). A full cycle of asexual development can be divided into five stages (Mims et al., 1988). The development of the conidiophore starts at a foot cell, which is a thicker part of the hyphae. A so-called stalk grows vertically out of the

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foot cell to a size of approximately 100 µm (Mims et al., 1988). A vesicle is formed at the tip of the stalk, which contains multiple nuclei. Proceeding from this vesicle, small mononuclear compartments, called metulae, are formed by cell budding. On the top of the metulae phialides are formed that in turn develop at their distal end conidiospores through mitotic divisions. The complete conidiophore is built 24 h after initiation of the foot cell structure (Bayram and Braus, 2012; Calvo et al., 2002; Mims et al., 1988; Timberlake, 1990). The airborne conidiospores are very resistant against environmental stressors and can be easily distributed. Their dark green cell wall pigmentation confers resistance against ultraviolet radiation (Adams et al., 1998;

Aramayo et al., 1989; Mayorga and Timberlake, 1990). Mature conidiospores can restart the fungal life cycle (Figure 7).

The sexual life cycle is promoted in the absence of light and under high carbon dioxide pressure.

The energy-consuming sexual life cycle results in the formation of a sexual fruiting body (cleistothecium), which is the overwintering structure of the fungus and can survive harsh environmental conditions (Braus et al., 2002; Pöggeler et al., 2018). Sexual development in A. nidulans starts with the fusion of two hyphae by wrapping of one hypha around the other. This results in lumpy hyphal structures (Casselton and Zolan, 2002; Sohn and Yoon, 2002). This nest-like structure, formed by hyphal fusion events, is surrounded by specialized cells. These so-called Hülle cells have nursing and protecting function for the maturing sexual fruiting body (Braus et al., 2002; Sarikaya-Bayram et al., 2010). Nest-like structures are observed approximately 24 h after initiation of the sexual life cycle (Figure 7). A primordium evolves in the following 24-48 h, which is the immature fruiting body and is characterized by a light reddish cell wall pigmentation (Brakhage, 1998). The immature fruiting body contains so-called ascogenous hyphae, which further develop a sac-like structure termed ascus (Greek: askos = sac): the name-giving structure of ascomycetes (Pöggeler et al., 2018).

The maturation of the cleistothecium is completed after seven days of development. The cell wall of the cleistothecium has a dark pigmentation and is surrounded by Hülle cells (Brown and Salvo, 1994; Sohn and Yoon, 2002). A meiotic nucleus division followed by a mitotic division inside the asci forms eight nuclei. Each nucleus is surrounded by a membrane. A subsequent mitotic nuclear division inside the small single compartments results in binucleate ascospores (Braus et al., 2002; Pöggeler et al., 2018). Bursting of a cleistothecium leads to release of thousands of ascospores that can easily be distributed into the environment. Each spore is able to initiate a new colony undergoing a new life cycle starting with the development of a complex network of vegetative hyphae.

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The development of multicellular structures in filamentous fungi like A. nidulans is concomitant with the production of certain secondary metabolites (Bayram and Braus, 2012). Secondary metabolites (SMs) are not essential for fungal growth but confer for example protective function or are important for intercellular communication (Brakhage, 2012). Secondary metabolites are responsible for the pigmentation of the cleistothecium and of the cell wall of the sexual and asexual derived spores (Adams et al., 1998; Brown and Salvo, 1994).

Figure 7: Life cycle of Aspergillus nidulans.

The development starts with a germinating spore, which forms a complex hyphal network.

Fungal mycelium reaches developmental competence and can respond to external or internal stimuli by initiation of multicellular development after 16-20 h of growth. Asexual development, resulting in formation of conidiophores, is favored in light, whereas the more energy-consuming formation of sexual fruiting bodies, called cleistothecia, is preferred in darkness and under high carbon dioxide pressure. Airborne conidiospores are formed by budding of spore forming cells (phialides) and as result of the asexual life cycle. Ascospores are formed inside the asci of cleistothecia. Conidio- and ascospores can re-initiate a fungal life cycle by germination and form vegetative hyphae.

SMs can be of great use for industrial or medical applications, but can also have toxic or carcinogenic impacts on microorganisms, plants and animals (including humans) (Yu and Keller, 2005). Penicillium chrysogenum and A. nidulans produce the antibacterial metabolite penicillin, which is one of the most useful secondary metabolites in clinical applications (Brakhage, 1998;

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Hemming, 1944). Other SMs like the family of aflatoxins produced by a number of Aspergilli, such as Aspergillus flavus, have toxic and carcinogenic effects on mammals (Yu, 2012). Inglis and co-workers annotated and identified 71 secondary metabolite gene clusters in A. nidulans, from which less than 20 are studied so far (Inglis et al., 2013). Most of these are silent under laboratory growth conditions, which makes the identification and characterization of their products challenging (Sanchez and Wang, 2013).