Multiple roles of a heterotrimeric G-protein gamma-subunit in governing growth and development of Aspergillus nidulans

Abstract

Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G-protein composed of FadA (G alpha), SfaD (G beta), and a presumed G gamma. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative G gamma-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (delta) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the delta sfaD mutant, the delta gpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively controlling FadA-mediated vegetative growth signaling were suppressed by delta gpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans G gamma-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression.

Figure 1.

Summary of GpgA structure. (A) A partial restriction map of a gpgA region is shown. The GpgA ORF (open box) and introns (discontinuities of the arrow) were determined by RT-PCR followed by sequencing. The solid box presents the G-protein gamma-like domain (GGL). (B) The steady-state gpgA mRNA levels in various growth and development stages of wild type (FGSC4) are shown. Numbers indicate incubation time (hours) in liquid submerged culture (Veg) or hours after developmental induction for asexual (Asex) or sexual (Sex) sporulation. Asc stands for ascospores (sexual spores). (C) Alignment of A. nidulans (An) GpgA with putative Gγ-subunits of Neurospora crassa (Nc; GNG-1, NCU00042.1), Gibberella zeae (Gz; XP_387411.1), and Saccharomyces cerevisiae (Sc; Ste18p, GI:6322545) is shown. Alignment was carried out via ClustalW with a default setting and displayed using BoxShade (identical and similar amino acids are in solid and shaded areas, respectively).

Figure 2.

Phenotypes of the mutant colonies. Relevant mutant and wild-type (WT) strains were point inoculated or streaked onto solid medium. The ΔgpgA mutant (RJAG19.9) exhibited a fluffy nondevelopmental phenotype for 2–3 days and then produced both conidiophores (CP) and Hülle cells (HC). The ΔgpgA mutant produced fewer asexual spores but more Hülle cells than the ΔsfaD (RSRB1.15) or ΔgpgAΔsfaD (RJA55.4) mutants. However, compared to WT, the ΔgpgA, ΔsfaD, and ΔgpgAΔsfaD mutants produced higher numbers of Hülle cells (HC). As evident in the close-up view of a single colony (SC) originated from a single conidium, the ΔgpgA mutant colonies do not produce conidiophores at this time (48 hr).

Figure 3.

Effects of G-protein mutations on vegetative growth and development. (A) Dry weights (percentage) of WT and G-protein mutant strains grown in liquid MM for 24 hr as well as numbers of (B) conidia (×10⁶/plate) and (C) Hülle cells (×10⁵/plate) produced by the colonies of designated strains grown on solid MM for 5 days are presented (average of triplicate cultures/measurements with standard error bars). Strains shown are WT (RJA56.25), Δγ (ΔgpgA; RJAG19.9), Δβ (ΔsfaD; RSRB1.15), Δα (ΔfadA; RJA71.4), and Δβγ (ΔsfaDΔgpgA; RJA55.4).

Figure 4.

Submerged conidiophore development caused by ΔsfaD but not by ΔgpgA. Conidiophore (CP) formation of WT and designated mutant strains grown in liquid submerged culture was observed from ∼17 to 36 hr at 1-hr intervals. Whereas ΔgpgA (RJAG19.9) or WT (RJA56.25) strains did not produce conidiophores even at 36 hr, ΔsfaD (RSRB1.15) and ΔgpgAΔsfaD (RJA55.4) strains began to elaborate conidiophores (CP) as early as 18 hr. The photographs were taken at 22 hr of growth in YM.

Figure 5.

Deletion of gpgA restores conidiation in the ΔflbA mutant. WT (RJA56.25), ΔgpgA (RJAG19.9), ΔsfaD (RSRB1.15), ΔflbA (RJA5.9), ΔgpgAΔflbA (RJA71.57), and ΔsfaDΔflbA (RSRF1.34) strains were point inoculated on solid MM and incubated at 37° for 3 days. Note that the ΔgpgAΔflbA and ΔsfaDΔflbA mutant colonies restored conidiation and no longer exhibited fluffy-autolytic phenotypes.

Figure 6.

Deletion of gpgA cannot bypass the need for fluG in conidiation. Designated strains were point inoculated on solid YM and incubated at 37° for 3 days. The ΔgpgAΔfluG (RJA41.18) and ΔsfaDΔfluG (RSR61.2) mutants were unable to produce conidiophores and formed fluffy colonies almost identical to those of the ΔfluG (RJA20.12) mutant.

Figure 7.

Genetic model for growth and developmental control in A. nidulans. We propose that the heterotrimer composed of FadA and SfaD∷GpgA functions in vegetative growth and fertilization signaling in A. nidulans (Yu et al. 1996; Rosén et al. 1999). Moreover, a recent study by Lafon et al. (2005; see accompanying article in this issue) revealed that GanB and SfaD∷GpgA constitute a functional heterotrimer that functions in conidial germination and sensing external carbon sources. In this model, it is speculated that during the vegetative growth phase FadA and SfaD∷GpgA primarily mediate signaling for proliferation. Activation of asexual/sexual development requires at least partial inhibition of this vegetative growth signaling. FlbA is an RGS protein that attenuates vegetative growth signaling by increasing the intrinsic GTPase activity of FadA (Lee and Adams 1994a; Yu et al. 1996). FluG activates asexual development by removing repressive effects imposed by multiple negative regulators (Seo et al. 2003), resulting in activation of a key transcription factor BrlA (see Adams et al. 1998). Genetic data suggest that GanB (Chang et al. 2004) and SfaD (Yu et al. 1996; Rosén et al. 1999) function in negative regulation of conidiation under submerged culture conditions. It is further speculated that FadA and SfaD∷GpgA may also function in signaling for sexual fruiting body development. A possible negative role of SfaD and/or GpgA in Hülle cell formation is indicated.