The velvet regulators

Important regulators of developmental programs in filamentous fungi are the velvet proteins, which form complex regulatory networks (Bayram et al., 2008a, 2008b; Käfer, 1965; Kato et al., 2003; Kim et al., 2002; Park et al., 2012a; Satterlee et al., 2016). Velvet proteins constitute a family of fungal specific regulatory proteins, which mostly comprises four members. The founding member of this family, velvet A (VeA), was identified more than half a century ago as a developmental regulator with a central role in transduction of the development inducing light signal (Käfer, 1965). The velvet family further comprises the factors VelB, VelC (velvet-like B and C) and VosA (viability of spores A). Velvet proteins share the name-giving velvet domain and are highly conserved among filamentous fungi

(Ahmed et al., 2013; Bayram et al., 2008a; Ni and Yu, 2007). The velvet domain does not exhibit sequence similarities with known protein domains, but structural similarities to the Rel homology domain of NF-κBs were found recently (Ahmed et al., 2013). NF-κBs constitute a family of mammalian transcription factors. They are involved in apoptosis and inflammatory response but also in broad metabolic processes and cell proliferation (Engelmann and Haenold, 2016; Sun and Andersson, 2002). Velvet factors contain a DNA-binding and dimerization domain and act as transcription factors in A. nidulans and Penicillium chrysogenum (Ahmed et al., 2013; Becker et al., 2016).

VeA is involved in the coordination of sexual development and secondary metabolism and is part of the light control of fungal development (Alkahyyat et al., 2015; Bayram et al., 2008a;

Calvo, 2008; Kim et al., 2002; Mooney and Yager, 1990; Stinnett et al., 2007). VeA is necessary for cleistothecia formation (Kim et al., 2002). Involvement of VeA and other velvet proteins in virulence has been shown in several fungi, such as A. flavus, several Fusarium spp.

and others (Duran et al., 2009; Merhej et al., 2012; Myung et al., 2012; Wang et al., 2016;

Wiemann et al., 2010). VeA interacts with several proteins. It forms a protein complex with the white-collar (WC) proteins LreA and LreB and the phytochrome FphA, which fulfils light-triggered regulatory functions (Hedtke et al., 2015; Purschwitz et al., 2008; Ruger-Herreros et al., 2011). WC proteins are involved in light regulation in fungi and activate expression of the major conidiation regulator-encoding bristle gene (brlA) in response to light (Chen et al., 2009; Froehlich et al., 2002; He and Liu, 2005; Ruger-Herreros et al., 2011;

Smith et al., 2010). VeA forms a heterotrimeric complex with VelB and the methyltransferase LaeA (lack of aflR expression A) in the nucleus, known as the velvet complex, which acts as a major regulator of secondary metabolism (Bayram et al., 2008a; Estiarte et al., 2016; Lind et al., 2016; Sarikaya-Bayram et al., 2010; Schumacher et al., 2015; Wang et al., 2016) (FIGURE 2) (see CHAPTER 1.3). The VeA-VelB heterodimer, which forms in the cytoplasm prior to velvet complex formation, is presumably the main mechanism for VelB to enter the nucleus as VelB does not exhibit a conserved nuclear localization sequence (NLS) (Bayram et al., 2008a; Bayram and Braus, 2012; Sarikaya-Bayram et al., 2010). Nuclear import of the VeA-VelB heterodimer is controlled by the methyltransferases VipC (VeA interacting protein C) and the VipC associated protein VapB (Sarikaya-Bayram et al., 2014). Both methyltransferases are recruited by VapA to the plasma membrane and released upon environmental triggers (Sarikaya-Bayram et al., 2014). The VipC-VapB heterodimer negatively influences VeA-VelB nuclear entrance after release from the plasma membrane. It also forms heterotrimeric complexes with VeA in the nucleus. Either VipC-VapB or the

heterotrimer acts positively on asexual and negatively on sexual development and influences histone posttranslational modifications (Sarikaya-Bayram et al., 2014, 2015) (FIGURE 2).

VelB was proposed to be an activator of conidiation since a loss of velB results in diminished conidiophores, whereas an overexpression (OE) leads to increased conidiation (Park et al., 2012b). VelB exhibits a positive regulation on the biosynthesis of sterigmatocystin, a potent mycotoxin (Bayram et al., 2008a; Bayram and Braus, 2012; Bryant et al., 2016; Gruber-Dorninger et al., 2016).

FIGURE 2: The velvet regulatory network.

The depicted schema summarizes the velvet protein network of A. nidulans. The α-importin KapA shuttles VeA-VelB into the nucleus. VipC-VapB is released from VapA at the plasma membrane and negatively regulates VeA-VelB nuclear entry. Both velvet proteins form several complexes in the nucleus. VeA-VelB recruits LaeA to form the velvet complex, which activates sexual development and secondary metabolism. VeA forms a heterotrimeric complex with VipC-VapB. Either this heterotrimer or VipC-VapB act as activator of asexual and repressor of sexual development and influence histone posttranslational modifications.

VelB forms homodimers and presumably acts positively on asexual development. The VelB-VosA heterodimer is important for spore viability and trehalose biosynthesis and acts as a repressor of early asexual development. The function of the VosA-VelC heterodimer is not clear, but it is proposed to positively regulate sexual development. Positive regulatory influences are shown in green, negative regulatory influences in red. Adapted from Sarikaya-Bayram et al., 2014, 2015.

VelB forms an alternative heterodimer with VosA in the nucleus (Sarikaya-Bayram et al., 2010) (FIGURE 2). The VelB-VosA heterodimer exhibits a time dependent dual function: it represses brlA expression during vegetative growth but regulates conidiospore viability and maturation by activation of wetA (wet-white A) and other genes, which products are important for conidiospore maturation and trehalose biosynthesis during late asexual growth (Bayram et al., 2008a; Lee et al., 2016; Ni and Yu, 2007; Park et al., 2012b; Sarikaya-Bayram et al., 2010) (see CHAPTER 1.5.3).

VelB and VosA, and their homologs, are inter-dependent in promoting spore maturation and viability (Sarikaya-Bayram et al., 2010; Wang et al., 2014; Webster and Sil, 2008). VosA is involved in conidiospore quality and virulence of several pathogenic fungi as well (Li et al., 2015; Wang et al., 2015). VeA and VosA seem to be exchanged as VelB binding partners in VelB heterodimers, since a deletion of laeA leads to increased VosA-VelB heterodimer formation (Sarikaya-Bayram et al., 2010).

The role of the fourth velvet protein VelC is a matter of ongoing investigation up to date.

In vitro analyses suggest the formation of a VosA-VelC heterodimer, which was proposed to positively regulate sexual development (Park et al., 2012a, 2014).