Regulation of septum formation by the Bud3-Rho4 GTPase module in Aspergillus nidulans

Abstract

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.

Figure 1.—

Effects of the Δbud3 deletion on colony morphology, septum formation, and conidiation. (A and B) Colony morphologies of strains TNO2A3 (wild type; A) and AHS3 (Δbud3; B) grown on minimal medium (MNUU) for 9 days. SepA–GFP localizes to hyphal tips, but not septa, in ΔAnbud3 mutants. (D) SepA–GFP localization at septa in wild-type hyphae. For C and D, ΔAnbud3 and wild-type strains possessing sepA∷gfp (AHS30 and AKS70, respectively) were grown in YGV media for 12 hr prior to imaging. (E) Wild-type conidiophore. (F) Δbud3 conidiophore. Fused metulae and phialides bearing a few spores were observed. (G and H) Wild-type (G) and Δbud3 (H) hyphae grown in YGVUU for 12 hr. Note the absence of septa in the Δbud3 mutant. Septa and nuclei were visualized in fixed hyphae using Calcofulor and Hoechst 33258, respectively. Arrows indicate septa. Bar, 10 μm.

Figure 2.—

Dosage suppression of Δbud3 growth and septation defects by rho4. (A–C) Colony morphology of Δbud3 strain AHS3 transformed with multiple copies of cdc42 (A), rac1 (B), or rho4 (C) and grown on selective MN media. Only rho4 functions as a dosage suppressor and restores conidiation. Hyphal morphology of Δbud3 strain AHS3 transformed with vector control (D) or rho4 (E). Hyphae were grown on YGV medium for 14 hr and stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. The presence of rho4 restored septum formation (arrows) to the ΔAnbud3 mutant. Bar, 10 μm.

Figure 3.—

Effects of the Δrho4 mutation on growth, hyphal morphology, and development. (A and B) Colony morphology of wild-type (TNO2A3; A) and Δrho4 (AHS5; B) strains following growth on MNUU medium for 9 days. (C and D) Hyphal morphology of wild-type (TNO2A3; C) and Δrho4 (AHS5; D) strains following growth for 14 hr on YGVUU at 28°. Arrows indicate septa. Bar, 10 μm. (E–G) Conidiophore morphology of wild-type (TNO2A3; E) and Δrho4 (AHS5; F–G) strains following growth for 3 days on MAGUU. Arrow in F indicates abnormal formation of a secondary conidiophore. Arrows in G indicate fused metulae and phialides. (H) Hyphal morphology of Δbud3 Δrho4 double mutant strain (AHS25) after 14 hr of growth in YGVUU at 28°. (I) Conidiophore morphology of Δbud3 Δrho4 double mutant strain (ASH25) following growth for 3 days on MAGUU. The arrow indicates abnormal formation of a secondary conidiophore generated from a phialide fused to its subtending metulae. Bars, 10 μm except for E–G, where bars = 3 μm.

Figure 4.—

AnBuD3 is a Rho4 exchange factor. In vitro guanine nucleotide exchange activity was determined by measuring binding of mant–GDP to purified Rho4 in the presence or absence of the putative GEF AnBud3 construct containing the GEF domain. The diagram indicates the mean values ± SD of at least two independent Rho protein and two GEF purifications with each experiment performed in duplicate. The intrinsic Rho activity is set to 100% (A). An example of in vitro kinetics for mant–GDP binding to purified AnRho4, AnBud3, and both is shown. The exchange activity of AnRho4 is stimulated by AnBud3 (B).

Figure 5.—

Localization of GFP–AnBud3. (A and B) GFP–AnBud3 rings (A, open arrows) and corresponding septa (B, solid arrows) following growth of strain AHS41 on YGVUU for 15 hr at 28°. The dashed arrow indicates a thick ring in the process of constricting. (C–J) Coordination of GFP–AnBud3 ring dynamics with septum deposition. (C, E, G, and I) GFP–AnBud3 localization. (D, F, H, and J) Calcofluor staining to visualize septa and cell walls. (C and D) GFP –AnBud3 localization at septation site prior to appearance of visible septum. (E and F) GFP–AnBud3 rings associated with thin septum. (G and H) Thicker AnBud3 ring associated with more prominent septum. (I and J) Constricting AnBud3 ring and associated septum. Bar, 3 μm, except A and B, where bar = 10 μm.

Figure 6.—

Localization of GFP–Rho4. (A and B) GFP–Rho4 localization (A) and corresponding septum (B; observed using DIC optics) following 13 hr growth of strain AHS43 at 28° on alcA(p) inducible threonine–MNV. Arrow indicates a GFP–Rho4 ring at the septation site. (C and D) A constricting GFP–Rho4 ring (C; white arrow) and corresponding septum (D; black arrow). Bar, 3 μm.

Figure 7.—

Recruitment of AnBud3 to septation sites does not require presence of the contractile actin ring. (A and B) GFP–AnBud3 localization in wild-type hyphae (AHS41) grown at 37° (A) or 28° (B). (C–H) GFP–AnBud3 localization in the sepA1 mutant (AHS51) at 37° (C, E, and G) and corresponding DIC images (D, F, and H). Dashed arrow (C) indicates a rare example of an intact GFP–AnBud3 ring, whereas solid arrows mark the more prevalent examples of incomplete rings or cortical patches. (I and J) GFP–AnBud3 localization (I) and corresponding DIC image (J) in sepA1 mutant hyphae 2 hr following a shift from 37° to 28°. Solid arrows indicate GFP–AnBud3 rings. Bars, 3 μm.

Figure 8.—

Localization of GFP–AnBud3 in the sepH 1 mutant. GFP–AnBud3 localization at 28° (A) and 42° (C) following 14-hr growth of strain AHS62 on YGV. GFP–AnBud3 localization to septation sites was not observed at 42°. B and D are corresponding DIC images. Arrows indicate septation sites. Bar, 5 μm.

Figure 9.—

Effects of the Δmsb1 mutation on growth and hyphal morphology. (A and B) Colony morphology of wild-type (TNO2A3; A) and Δmsb1 (AHS7; B) strains following growth on MNVUU medium for 6 days and 7 days, respectively. (C and D) Hyphal morphology of wild-type (TNO2A3; C) and Δmsb1 (AHS7; D) strains following growth for 13 hr on MNVUU at 28°. Arrows indicate septa. Bar, 10 μm.