ZtfA is a repression target of VosA and acts as a conidiation regulator in A. nidulans

4.2.1 The upstream developmental activator pathway is regulated by ZtfA

Velvet proteins are transcription factors, which interconnect developmental programs and secondary metabolism and regulate expression of thousands of genes (Ahmed et al., 2013;

Becker et al., 2016). The velvet factor VosA represses premature conidiation and is important for spore viability (Ni and Yu, 2007). The present study shows that ZtfA is a downstream target of VosA. VosA is a repressor of ztfA gene expression and both factors together are important for viability of conidiospores.

VosA represses expression of the major conidiation regulator-encoding brlA gene during vegetative growth and therefore regulates developmental competence (Lee et al., 2016; Ni and Yu, 2007). BrlA is the first factor in the central developmental pathway (CDP) (BrlA  AbaA  WetA) and is activated by the upstream developmental activators (UDAs) (Adams et al., 1998; Lee and Adams, 1996; Lee et al., 2016; Marshall and Timberlake, 1991;

Mirabito et al., 1989). The UDAs are repressed by SfgA and comprise the Flb pathway, which functions in two parts in parallel: FlbB/FlbE  FlbD  BrlA and FlbC  BrlA (Lee et al., 2016; Ruger-Herreros et al., 2011; Seo et al., 2006; Yu et al., 2010). FluG counteracts the repressing effect of SfgA, what allows activation of the Flbs (Garzia et al., 2010; Kwon et al., 2010a, 2010b; Ni and Yu, 2007; Seo et al., 2006; Wieser and Adams, 1995).

It was previously hypothesized, that another yet unknown factor might exist that regulates conidiation together with the Flb cascades (Garzia et al., 2010; Lee and Adams, 1996). Such a factor was anticipated to activate brlA gene expression or expression of either flb factors in the last steps of the UDA pathway, namely flbC and flbD (FIGURE 51). flb gene knock outs share the name-giving phenotype: a fluffy appearance of the colony due to increased aerial hyphae and decreased conidiophore production (Wieser et al., 1994).

Phenotypical and transcriptional analyses show that ZtfA functions downstream of FluG and upstream of FlbC and FlbD. It is not involved in the regulation of sfgA expression but is an activator of flbC and flbD gene expression during late vegetative growth. Taken together,

ZtfA represents a new activator of conidiation, which functions upstream to the UDA pathway and is important for activation of both parts of the Flb cascade (FIGURE 51).

FIGURE 51: Gene expression of flbC, flbD and brlA is activated by ZtfA.

The model summarizes the current understanding of the regulatory role of ZtfA upon the UDA factors and brlA during late vegetative growth at the onset of conidiation. ZtfA activates brlA gene expression and is important for activation of FlbC and FlbD, downstream of FluG.

Positive regulations are given in black, negative regulations (repression) are given in red.

4.2.2 ZtfA activates brlA expression in A. nidulans

ZtfA is sufficient to induce premature conidiophore development during vegetative growth and activates brlA expression under these conditions. This is further supported by the finding that this activation is significantly higher in the absence of vosA. VosA and NsdD repress brlA expression during vegetative growth (Lee et al., 2016; Ni and Yu, 2007). ztfA OE produces normal conidiophores during these conditions, whereas the WT is not able to form conidiophores. Overexpression of brlA leads to conidiophore formation under vegetative conditions directly from vesicles formed at the hyphal tips (Adams et al., 1988, 1998).

Therefore, several aspects of the regulatory role of ZtfA are important for the formation of conidiophores, besides its activating role upon brlA. ZtfA regulates flbD expression and is important for flbC expression during vegetative growth. However, this is not sufficient to induce conidiophore development, since a flbC OE does not form conidiophores but vesicle-like structures at hyphal tips (Kwon et al., 2010a). Therefore, induction of gene expression of flbC, flbD and brlA together is necessary for the conidiophore formation. BrlA activates abaA and wetA, which both encode transcription factors necessary for conidiation (Andrianopoulos and Timberlake, 1994; Lee et al., 2016; Marshall and Timberlake, 1991; Ni and Yu, 2007;

Sewall et al., 1990a). In accordance with the finding that ZtfA activates brlA, phenotypical analyses show that ztfA is epistatic to both, brlA and abaA. Taken together, ZtfA activates both, the UDA factors through flbC and flbD and the CDP through brlA.

4.2.3 ZtfA and VosA function in achievement of developmental competence and spore maturation in A. nidulans

The onset of conidiation is genetically characterized by a time dependent de-repression of brlA expression (see CHAPTER 1.4 and 1.5) (Lee et al., 2016). The conidiation cascade itself is also time dependent: brlA is activated by the UDA factors and ZtfA during the achievement of developmental competence (Etxebeste et al., 2009, 2008; Garzia et al., 2009, 2010; Kwon et al., 2010a, 2010b; Wieser and Adams, 1995) (FIGURE 52).

FIGURE 52: ZtfA and VosA regulate achievement of developmental competence and spore maturation.

The model depicts the regulatory roles of ZtfA and VosA upon conidiation during achievement of developmental competence (upper part) and ongoing spore formation and maturation (lower part). ZtfA activates gene expression of brlA, flbC and flbD during late vegetative growth and supports achievement of developmental competence. brlA expression is repressed by VosA and NsdD during this stage (upper part). ZtfA negatively regulates fluG and flb gene expression during ongoing development and supports spore maturation. vosA expression is activated by AbaA and WetA and VosA is necessary for trehalose biogenesis and spore maturation as well. VosA negatively regulates ztfA expression during asexual growth (lower part).

BrlA activates abaA expression during the mid-phase of conidiation (approximately 12 h post induction), whose product then activates wetA and vosA expression during the late phase of

conidiation (after 24 h post induction) (Adams et al., 1988, 1990; Andrianopoulos and Timberlake, 1994; Boylan et al., 1987; Marshall and Timberlake, 1991; Ni and Yu, 2007; Tao and Yu, 2011). VosA is involved in early time tuning of conidiation by repressing brlA expression until developmental competence is achieved (Lee et al., 2016; Ni and Yu, 2007) (FIGURE 52). During late asexual growth, VosA negatively regulates ztfA expression, thereby exhibiting another level of regulation of the time adjusted choreography of conidiation. ZtfA is involved in late time tuning of the conidiation cascade as well: it negatively regulates fluG and the flb genes during late asexual growth after 24 h (FIGURE 52). This regulatory role confirms the model that ZtfA functions upstream of the UDA pathway as a rather global regulator of conidiation.

AbaA induces vosA expression during late asexual development (Park et al., 2012b) (FIGURE 52). VosA is necessary for spore viability and trehalose biogenesis together with VelB (Ni and Yu, 2007; Sarikaya-Bayram et al., 2010). Trehalose is a non-reducing disaccharide that is found in fungi, plants, bacteria and insects, where it functions as storage compound, but is also involved in stress resistance (Becker et al., 1996; Elbein et al., 2003;

Jorge et al., 1997; Ocón et al., 2007). In germinating conidiospores, trehalose is rapidly degraded and deletion of the trehalose-6-phosphate synthase results in delayed germination (Al-Bader et al., 2010; Fillinger et al., 2001; Kane and Roth, 1974; Shin et al., 2009).

Conidiospores are able to germinate in the absence of ztfA, but show a rapid loss in viability.

Decreased spore viability has been shown before for vosA and velB mutants and for other A. nidulans mutant strains as well (Hagiwara et al., 2008; Kawasaki et al., 2002; Lara-Rojas et al., 2011; Leiter et al., 2016). Trehalose has been shown to be important for conidiospore viability: a loss in spore viability accompanies an insufficient trehalose concentration in conidiospores in ΔvosA and ΔvelB strains (Ni and Yu, 2007; Sarikaya-Bayram et al., 2010).

Loss in spore viability in ΔztfA is not coupled to trehalose biogenesis and ZtfA is not important for regulation of trehalose biosynthesis. Hence, ZtfA is not involved in the governed trehalose biogenesis regulation. Nevertheless, ZtfA probably is a factor of a VosA-governed spore viability regulation. ZtfA’s role in spore viability, together with the activation of brlA and flb genes during late vegetative growth, supports the model that ZtfA is involved in the activation of conidiation and its regulation during ongoing spore formation and maturation. Such a regulation is probably not based upon VeA-VelB or VelB-VosA heterodimers since phenotypic analyses with ΔveAΔztfA and ΔvelBΔztfA double mutants do not imply relations between these velvet genes and ztfA. A VosA-VelC heterodimer has been proposed but has not been proven in vivo up to date (Park et al., 2014). Since little is known

about VelC at the moment, a regulatory effect upon spore viability cannot be excluded. A ΔvelCΔztfA double mutant does not show clear epistatic effects. Therefore, the regulatory effects of ZtfA are probably not dependent upon VelC.