Regulation of Septum Formation by the Bud3â•fiRho4GTPase Module in Aspergillus nidulans

The ability of fungi to generate polarized cells with a variety of shapes likely reflects precise temporal and spatial control over the formation of polarity axes. The bud site selection system of Saccharomyces cerevisiae represents the best-understood example of such a morphogenetic regulatory system. However, the extent to which this system is conserved in the highly polarized filamentous fungi remains unknown. Here, we describe the functional characterization and localization of the Aspergillus nidulans homolog of the axial bud site marker Bud3. Our results show that AnBud3 is not required for polarized hyphal growth per se, but is involved in septum formation. In particular, our genetic and biochemical evidence implicates AnBud3 as a guanine nucleotide exchange factor for the GTPase Rho4. Additional results suggest that the AnBud3–Rho4 module acts downstream of the septation initiation network to mediate recruitment of the formin SepA to the site of contractile actin ring assembly. Our observations provide new insight into the signaling pathways that regulate septum formation in filamentous fungi. THE filamentous fungi form mycelial colonies that consist of networks of branched hyphae that grow by apical extension. In the higher fungi (i.e., Ascomycota and Basidiomycota), hyphae are compartmentalized by the formation of cross-walls, or septa. It has long been suspected that the presence of septa allows filamentous fungi to partition cellular environments within a hypha to support colony homeostasis and reproductive development (Gull 1978). The process of septum formation is similar to cytokinesis of animal cells, in that it coordinated with mitosis and requires formation of a contractile actin ring (CAR) (Balasubramanian et al. 2004). By analogy to the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the CAR likely provides a landmark that guides deposition of the septal wall material. However, unlike these yeasts, the septum is not subsequently degraded and cells remain attached. Furthermore, in most filamentous fungi, a small pore is retained to enable communication between adjacent hyphal compartments. Septum formation has been studied in several filamentous fungi, including Aspergillus nidulans (Harris 2001; Walther and Wendland 2003). Upon germination of asexual conidiospores in A. nidulans, the first few rounds of parasynchronous nuclear division are not accompanied by septation until cells reach an appropriate size/ volume (Harris et al. 1994; Wolkow et al. 1996). Subsequently, the first septum forms near the junction of the spore and germ tube (Harris et al. 1994). Deposition of the septal wall material is tightly coupled to assembly and constriction of the CAR, which in turn requires persistent signals from mitotic nuclei (Momany and Hamer 1997). As A. nidulans hyphae continue to grow by apical extension, each parasynchronous round of mitosis in multinucleate tip cells is followed by formation of septa in the basal region of the compartment (Clutterbuck 1970). Because tip and intercalary hyphal cells are multinucleate, not all of the individual mitotic events within the tip cell are capable of triggering septation. Genetic analyses have identified several functions required for septum formation in A. nidulans, including the septation initiation network (SIN), the septins, and a formin (Harris 2001). The SIN is a cascade of three protein kinases that is activated by a small GTPase (Krapp and Simanis 2008). In A. nidulans, the component kinases of the SIN are arranged in the pathway SepH/SepL/SidB, with SepM amd MobA serving as cofactors that regulate SepL and SidB, respectively (Kim et al. 2006, 2009). Although SIN components localize to the spindle pole bodies, this does not appear to be a prerequisite for their subsequent recruitment to the septation site (Kim et al. 2009). Functional analysis of SepH, ModA, and SidB demonstrate that the SIN is required for assembly of the CAR (Bruno et al. 2001; Kim et al. 2006). Nevertheless, the upstream activators of Supporting information is available online at http:/ www.genetics.org/ cgi/content/full/genetics.110.114165/DC1. Corresponding author: Center for Plant Science Innovation, E126 Beadle Center, University of Nebraska, Lincoln, NE 68588-0660. E-mail: [email protected] Genetics 185: 165–176 (May 2010) the SIN and its downstream effectors remain unknown. However, localization of the septin AspB and the formin SepA to the septation site have been shown to require SepH (Sharpless and Harris 2002; Westfall and Momany 2002). AspB initially appears as a single ring that does not constrict, but splits into a double ring flanking the septum (Westfall and Momany 2002). AspB is not required per se for assembly of the CAR (Westfall and Momany 2002). On the other hand, SepA is a dynamic component of the CAR that is required for its assembly (Sharpless and Harris 2002), presumably because of its ability to nucleate actin filaments. In S. cerevisiae and S. pombe, formins such as SepA are typically activated by Rho GTPases, such as Rho1 and Cdc42 (e.g., Dong et al. 2003; Martin et al. 2007). However, neither Cdc42 nor Rac1 is required for septum formation in A. nidulans, and Cdc42 does not localize to septation sites (Virag et al. 2007). One promising candidate for a GTPase that could activate SepA is Rho4, which appears to be specific to filamentous fungi (Rasmussen and Glass 2005). In Neurospora crassa, Rho4 is a dynamic component of the CAR; its absence prevents CAR assembly, whereas constitutive activation permits spurious formation of extra CARs (Rasmussen and Glass 2005). On the basis of these results, it was suggested that Rho4 is a likely activator of formins such as SepA at septation sites. Because SepA simultaneously localizes to hyphal tips and septation sites in A. nidulans (Sharpless and Harris 2002), we have been interested in the identification of functions that determine patterns of cell wall deposition in hyphal cells. In this context the bud site selection system of S. cerevisiae provides an important paradigm. S. cerevisiae cells display two distinct budding patterns that are controlled by mating type (Freifelder 1960; Chant 1999). Mating type a or a cells employ an axial budding pattern whereby the previous bud site serves as a template for the next bud. As a result, a chain of chitinous bud scars decorates the cell surface. In contrast, mating type a/a cells employ a bipolar budding pattern whereby buds emerge from either the distal or proximal pole of the cell (the proximal pole is defined by the presence of the birth scar, Chant and Pringle 1995). Accordingly, bud scars cluster at either pole but are not necessarily adjacent to each other. Extensive genetic analyses have provided a fairly detailed understanding of the molecular mechanisms that underlie the axial and bipolar budding patterns. For the axial pattern, the cell wall protein Axl2 serves as a landmark whose function is facilitated by its association with Axl1 and the septin-interacting proteins Bud3 and Bud4 (Chant and Herskowitz 1991; Chant et al. 1995; Chant 1999; Lord et al. 2002; Gao et al. 2007; Park and Bi 2007). For the bipolar pattern, the paralogous cell wall proteins Bud8 and Bud9, which bear no homology to Axl2, serve as distal and proximal pole markers, respectively (Chant 1999; Harkins et al. 2001; Kang et al. 2004; Park and Bi 2007). Furthermore, the membrane proteins Rax1 and Rax2 form complexes with Bud8 and Bud9, which facilitates their function (Kang et al. 2004). The positional information generated by the landmark proteins Axl2, Bud8, or Bud9 is subsequently relayed to the Ras-like Bud1/Rsr1 GTPase module via the guanine nucleotide exchange (GEF) factor Bud5 (Kang et al. 2001, 2004; Krappmann et al. 2007). This results in localized activation of the Rho-like GTPase Cdc42, which acts via multiple effectors to recruit components of the morphogenetic machinery to the specified bud site (Chant 1999; Park and Bi 2007). Despite the importance of the bud site selection regulatory module in specifying the budding pattern of S. cerevisiae yeast cells, it remains unclear whether it is used for a similar regulatory purpose in other fungi. Ashbya gossypii is a hemiascomycete fungus closely related to S. cerevisiae that is only capable of forming hyphae (Philippsen et al. 2005). The A. gossypii Bud3 homolog, which can function in S. cerevisiae, appears to function as a landmark for septum formation and also controls the position of the contractile actin ring (Wendland 2003). In A. gossypii and Candida albicans, another hemiascomycete capable of forming true hyphae, Bud1/Rsr1 homologs appear to function at the hyphal tip to specify the direction of hyphal extension (Bauer et al. 2004; Hausauer et al. 2005). Although limited to hemiascomycetes, these studies suggest that the components of the bud site selection regulatory module may have a broader function within the fungal kingdom. Here, we investigate the possibility that homologs of the bud site selection proteins may provide positional information that marks the hyphal tip and/or septation sites in A. nidulans. We characterize an apparent homolog of Bud3 and show that it is required for assembly of the CAR at septation sites. Our results provide new insight into the regulation of septum formation by suggesting that AnBud3 functions downstream of the SIN as a GEF for Rho4. MATERIALS AND METHODS Strains, media, growth conditions, and staining: Aspergillus nidulans strains used in this study are listed in Table 1. Minimal 1 vitamins (MNV) media were made according to Kafer (1977). MNV-glycerol and MNV-threonine fructose media were made as described in Pearson et al. (2004). Malt extract agar (MAG) and yeast extract glucose 1 vitamins (YGV) media were made as described previously (Harris et al. 1994). The reagent 5-fluoroorotic acid (5-FOA; US Biological, Swampscott, MA) was added to media at a concentration of 1 mg/ml after autoclaving. For septation and hyphal growth studies, conidia from appropriate stains were grown at 28 for 12 hr on cover slips. Hyphae attached to the cover slip were fixed using a modified standard protocol (Harris et al. 1994) [fixing solution contained 3.7% formaldehyde, 25 mm EGTA, 50 mm 166 H. Si et al.