Regulation of fungal development

A. nidulans is a soil borne fungus and its life is, like in most other organisms, regulated by light. Light sensing is crucial to adapt to changing environmental conditions. For spreading hyphae it is a major difference whether they grow on or under the surface in terms of abiotic and biotic factors such as illumination, oxygen concentration, osmotic stress and reactive oxygen species (Rodríguez-Romero et al, 2010; Bayram & Braus, 2012; Jaimes-Arroyo et al, 2015). The choice of entering the asexual or sexual developmental program is not a black/white decision. Light predominantly induces asexual (conidiophore) development and represses sexual (cleistothecia) development. After competence is acquired, induction of spore formation occurs through intracellular programmed events, which include the

A. nidulans senses light with a variety of different light receptors. The fungal phytochrome FphA is a sensor kinase that represses sexual development and induces asexual spore formation under red-light conditions (Blumenstein et al, 2005; Brandt et al, 2008). Phytochromes exist in two different conformations that can change upon light perception of a certain wavelength. The ratio between the two forms determines the signaling state of the phytochrome (Bayram et al, 2010). Cryptochromes are blue/UV light receptors that presumably originated from the DNA photolyase protein family, which are light-activated DNA-repair enzymes (Lin & Todo, 2005). Blue-light receptors of A. nidulans are encoded by lreA (Blue-light receptor A) and lreB. These transcription factors are homolog to the white-collar proteins of N. crassa WC1/WC2 that regulate the circadian rhythm. LreA and LreB stimulate sexual development and are associated to FphA (Purschwitz et al, 2008; Chen et al, 2010; Bayram & Braus, 2012; Dasgupta et al, 2015). The nuclear photolyase-like protein CryA (Cryptochrome A) senses UV-light and represses sexual development. Deletion of cryA results in abnormal formation of Hülle cells in submerged culture and increased cleistothecia formation during UV or blue-light illumination (Bayram et al, 2008a;

Dasgupta et al, 2015).

A key protein involved in the regulation of light dependent developmental is encoded by veA. The velvet protein VeA is a conserved transcription factor that is supposed to affect the expression of hundreds of genes and physically interacts with FphA (Purschwitz et al, 2008; Dhingra et al, 2012; Sarikaya-Bayram et al, 2015).

Deletion of veA results in a block of sexual development and reduces the production of secondary metabolites such as the aflatoxin precursor sterigmatocystin and the antibiotic penicillin (Kato et al, 2003; Bayram & Braus, 2012; Gerke & Braus, 2014).

Light controls cellular localization of VeA. During illumination, VeA is associated to the velvet-like protein VelB and predominantly localized in the cytoplasm (Figure 10). In darkness the two velvet family proteins VeA-VelB interact with importin KapA (karyopherin A) to enter the nucleus (Stinnett et al, 2007; Bayram et al, 2008b). The nuclear VeA-VelB heterodimer forms the trimeric velvet complex by binding the histone methyltransferase LaeA (lack of aflR expression A), which is a master regulator of secondary metabolism (Sarikaya-Bayram et al, 2010). The trimeric velvet complex VelB-VeA-LaeA is essential to coordinate secondary metabolism and development by inducing the expression of sexual as well as

INTRODUCTION

Beside LaeA, two other methyltransferases namely VipC (VeA interacting protein C) and VapB (VipC associated protein B) interact with VeA. The VipC-VapB dimer forms a complex with the membrane protein VapA (VipC associated protein A) to allow the nuclear velvet complex VelB-VeA-LaeA to induce transcription of genes important for sexual development in the dark. The release of membrane bound VipC-VapB by external signals results in VeA interaction, leading to a reduced import of the VeA-VelB dimer into the nucleus (Sarikaya-Bayram et al, 2014; 2015).

VelB might be part of a second complex as it interacts with the transcription factor VosA in the nucleus. The VelB-VosA heterodimer regulates the expression of genes important for cell wall biosynthesis and is required for spore viability and repression of asexual development in the dark (Sarikaya-Bayram et al, 2010; Park et al, 2015).

Asexual development of A. nidulans is a precisely timed and genetically coordinated process, in which light regulates expression of several hundred genes in developmentally competent mycelium (Ruger-Herreros et al, 2011). The central regulatory pathway is composed of the genes brlA, abaA and wetA (Figure 10).

Respective proteins control asexual development-specific genes and determine the order of gene expression during conidiation (Mirabito et al, 1989; Timberlake, 1990;

Adams et al, 1998). The master regulator BrlA, whose transcription is induced by upstream regulators such as FlbB, is a transcription factor that is expressed in early asexual development when the conidiophore vesicles start to form (Momany, 2015).

BrlA is localized in vesicles, metulae and phialides. Deletion of brlA leads to fungal colonies with elongated stalks, unable to develop vesicles or any other subsequent structures, thus the respective phenotype was named “bristle” (Clutterbuck, 1969a).

BrlA is required for the expression of developmentally regulated genes (Boylan et al, 1987). Expression of abaA depends on BrlA activity and is initiated during phialide formation (Yu, 2010). Mutants deleted for abaA produce aconidial conidiophores that develop sterigmata but are incapable to form sporogonous phialides. Therefore, deletion of abaA results in the repetition of phialide-like structures rather than chains of conidia (Sewall et al, 1990b). brlA is overexpressed in abaA mutants, but abaA overexpression results in expression of wetA and brlA, demonstrating that AbaA has suppressive as well as promoting activities towards brlA (Aguirre, 1993; Sewall et al, 1990b). The third protein participating in the central regulatory pathway of asexual

activating the wetA gene, indicating an autoregulatory expression mechanism (Boylan et al, 1987). WetA activates genes required for spore formation and maturation (Sewall et al, 1990a). Other light induced genes contribute to spore resistance in a dry environment (Suzuki et al, 2013).

Figure 10: Light-dependent regulation of A. nidulans development.

In darkness, the VeA-VelB dimer interacts with importin KapA to enter the nucleus through the nuclear pore (NP). VapA binds the methyltransferases VipC and VapB to the membrane. Light as well as the membrane unbound VipC-VapB dimer repress nuclear import of the VeA-VelB complex. In the nucleus, VeA-VelB can interact with the methyltransferase LaeA to form the trimeric velvet complex, which coordinates sexual development and secondary metabolism. In addition, VelB forms a dimer with VosA that inhibits asexual development in the dark and is crucial for spore viability by activating trehalose biogenesis. Light decreases cellular levels of VosA and VelB and promotes asexual development, which is controlled by a central regulatory pathway. BrlA and the downstream transcription factors AbaA and WetA constitute the central regulatory pathway of conidiation. Increased brlA expression is crucial for conidiophore vesicle formation and conidiophore maturation. BrlA activates expression of abaA and wetA. AbaA is required for phialide differentiation and has activating as well as repressing functions towards brlA. abaA induces expression of wetA, which has an autoregulatory expression mechanism and is crucial for spore maturation. Modified from (Bayram & Braus, 2012; Sarikaya-Bayram et al, 2014).

INTRODUCTION

An additional key player to complete asexual development in A. nidulans is VosA that also interacts with VelB in response to light. The VosA encoding gene is expressed during the formation of both sexual and asexual spores. VosA is conserved in most fungi and controls the late phase of conidiation. VosA is required for activating genes involved in spore maturation, but represses certain development-specific genes (Ni & Yu, 2007; Park et al, 2015). Strains deleted for vosA reveal a loss of trehalose accumulation in spores that is crucial to protect cells against environmental stresses (Ni & Yu, 2007).

1.6.3 The deneddylases COP9 signalosome and DEN1 in Aspergillus nidulans