ZtfA is a regulator of conidiation and secondary metabolism in A. nidulans

Members of the genus Aspergillus are among the most widespread fungi worldwide and colonize diverse ecological niches. Most of these filamentous fungi are saprophytic and important for biological substance cycles. A number of Aspergilli are secondary metabolite producers and several Aspergilli have deleterious as well as beneficial effects on humankind.

Secondary metabolism is linked to developmental programs in filamentous fungi through the velvet proteins. This study characterizes a novel target of the velvet factor VosA: the transcription factor ZtfA. ztfA gene expression is repressed by VosA and both factors are necessary for spore viability. ZtfA activates conidiophore formation by expression regulation of the major conidiation activator-encoding brlA gene and its upstream activators, encoded by flbC and flbD. Moreover, ZtfA regulates biosynthesis of several secondary metabolites through activation of expression of their gene clusters. Besides, ZtfA is important for an appropriate response towards oxidative stress. It acts as activator of the thioredoxin system and catA gene expression, but represses other factors of the OSR. A summarizing model of the regulatory influences of ZtfA is shown in Figure 50.

FIGURE 50: Comprehensive model of the regulatory role of ZtfA in A. nidulans.

The model describes the major regulatory roles of ZtfA as activator of conidiation and secondary metabolism and regulator of the oxidative stress response. Green arrows indicate positive and red lines negative influences on gene expression.

4.1.1 The C6 domain of ZtfA is highly conserved in Aspergilli

Proteins with a C6 domain constitute a group of fungal specific DNA binding proteins, which act as transcription factors (Chang and Ehrlich, 2013; MacPherson et al., 2006; Schjerling and Holmberg, 1996). The C6 domain of ZtfA deviates from the domains of most common zinc cluster proteins in A. nidulans. Wortman and collaborators identified 330 C6 proteins in A. nidulans (Wortman et al., 2009). An up-to-date in silico search, employing the fungal databases FungiDB and AspGD (Cerqueira et al., 2014; Stajich et al., 2012) reveals a recent number of 332 putative C6 proteins in A. nidulans. Less than six percent of all C6 proteins in A. nidulans exhibit a CX2CX6CX5CX2CX8C domain architecture, amongst them ZtfA. Only two other C6 proteins with this architecture have been characterized in A. nidulans so far:

AcuM and ClrB (Coradetti et al., 2012; Hynes et al., 2007). Both are involved in primary metabolism: AcuM is involved in gluconeogenesis, whereas ClrB was identified as a regulator of cellulase gene expression. Its uncommon C6 architecture together with its broad regulatory influences renders ZtfA a very unique protein among C6 proteins in A. nidulans.

The C6 domain of the yeast DNA-binding protein Gal4, the best-studied C6 protein, has a CX₂CX₆CX₆CX₂CX₆C architecture (Giniger et al., 1985; Marmorstein et al., 1992; Pan and Coleman, 1990; Rodgers and Coleman, 1994). This is the most common C6 architecture in A. flavus and A. nidulans, followed by CX₂CX₆CX₅CX₂CX₆C in a ratio of 2:1 (Chang and Ehrlich, 2013). In general, the cysteines within the first part of this motif are conserved whereas the second part varies and forms different architectures: CX₂CX₆CX_5-16CX₂CX_6-8C (Todd and Andrianopoulos, 1997). The databank searches conducted in this study as well as the data published by Wortman and collaborators reveal that the last part has to be extended for A. nidulans to CX₂CX₆CX_5-16CX₂CX_4-12C and that few exceptions from this general architecture exist. An overview of the different architectures present in A. nidulans and characterized proteins exhibiting these architectures is given in TABLE 10.

The C6 domain is required for DNA binding (Bai and Kohlhaw, 1991; Burger et al., 1991;

Defranoux et al., 1994; Johnston and Dover, 1987; Pfeifer et al., 1989; Todd et al., 1997;

Todd and Andrianopoulos, 1997). Amino acids within the first CX2CX6C motif are conserved and mutagenesis studies showed their importance for DNA binding (Johnston and Dover, 1987; Todd and Andrianopoulos, 1997; Yuan et al., 1991).

DNA recognition sites consisting of terminal trinucleotides of direct or inverted repeats, which are separated by six to eleven nucleotide residues, have been characterized for several C6 proteins. CCG triplets as everted or inverted repeats have been proposed to be typical C6 consensus sequences (MacPherson et al., 2006; Marmorstein et al., 1992). AcuM binds a

CCGN₇CCG nucleotide consensus sequence, presumably as a heterodimer (Suzuki et al., 2012). ClrB was shown to bind to CGGN₈CCG inverted repeats in a co-factor dependent manner, as well as to CGG/CCG single triplets in the absence of a co-factor (de Groot et al., 2009; Li et al., 2016; Yamakawa et al., 2013). However, binding to triplets of different structure has been shown for other C6 proteins as well (Chang et al., 1995; Todd et al., 1998;

Todd and Andrianopoulos, 1997).

TABLE 10: Comprehensive overview of C6 architectures present in A. nidulans.

The table provides an overview over the frequency of C6 architectures in A. nidulans, according to Wortman and collaborators, amended with the two additional proteins found in AspGD and FungiDB database searches (Cerqueira et al., 2014; Stajich et al., 2012; Wortman et al., 2009). All characterized representatives of each group are indicated. ZtfA and its architectural group are given in bold.

C6 motif Quantity Metabolic processes Development Stress response

CX₂CX₆CX₆CX₂CX₆C 149 AflR, DbaA, MdpE, ArcA,

Several conserved amino acid residues are present in ZtfA giving a hint that ZtfA might recognize nucleotide triplet repeats as well. In Gal4, the lysine residues K17 and K18 were identified to be necessary for the specific base pair contact to the CGG triplet forming the DNA binding motif (Marmorstein et al., 1992; Marmorstein and Harrison, 1994). RhaR is an example for a C6 protein in A. nidulans, which shows conservation of K18 but replacement of lysine K17 to arginine (R68) (Pardo and Orejas, 2014). RhaR binds to a CGGN11GGC DNA motif. ZtfA exhibits a stretch of three lysines at this position (K4-6). The relatively uncommon C6 architecture of ZtfA and indications for different interaction partners suggest that ZtfA is able to bind different DNA-binding sequences. Importantly, among ZtfA orthologs in Aspergilli the whole C6 domain of ZtfA is strongly conserved. These similarities in ZtfA orthologs imply that it might bind to similar binding motifs in different Aspergilli. It is therefore assumed, that ZtfA regulates the expression of orthologous genes and exhibits influences similar to the ones found for A. nidulans in other Aspergilli as well.

4.1.2 C6 proteins and their role in A. nidulans

The A. nidulans genome encodes 332 putative C6 proteins. In contrast to this high number, which makes up approximately three percent of all genes in A. nidulans, a relatively small number of only 33 C6 proteins (approximately 10%) has been characterized yet (TABLE 10).

Several proteins of this group regulate primary or secondary metabolite gene clusters as cluster specific regulators in A. nidulans, such as AmdR, AlcR, QutA, NirA, PrnA, UaY, FacB, MdpE and AflR (Andrianopoulos and Hynes, 1990; Bergmann et al., 2007; Beri et al., 1987; Brown et al., 1996; Burger et al., 1991; Cazelle et al., 1998; Chiang et al., 2009, 2010;

Felenbok et al., 1988; Scazzocchio, 1994; Suarez et al., 1995; Todd et al., 1997). Two C6 proteins, SfgA and OefC, are involved in the regulation of asexual sporulation, and three C6 proteins, RosA, NosA and SilA, were found to be involved in sexual development (Han et al., 2008; Lee et al., 2005; Seo et al., 2003, 2006; Vienken et al., 2005; Vienken and Fischer, 2006). Employment of the AspGD Gene Onthology slim mapper (Cerqueira et al., 2014) showed, that most manually annotated C6 proteins cluster with the terms carbohydrate metabolic processes, developmental processes and response to chemical or stress. Three are annotated to secondary metabolic processes, three for sexual sporulation and two for asexual sporulation. So far, most of the C6 proteins analyzed for A. nidulans regulate metabolic processes as cluster specific transcription factors or are involved in developmental programs.

Regulation and interconnection of several processes and programs on a higher level, as this study shows for ZtfA, is uncommon for C6 proteins in A. nidulans. As only 10% of this

protein group has been investigated so far, it is likely to find more proteins which interconnect different pathways in this transcription factor group in future analyses.

In conclusion, ZtfA is one of the first C6 proteins that act as activator for asexual development and secondary metabolism.

4.2 ZtfA is a repression target of VosA and acts as a conidiation regulator in