The heterotrimeric G-protein GanB(alpha)-SfaD(beta)-GpgA(gamma) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillus nidulans

Abstract

The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the G alpha-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB(alpha)-SfaD(beta)-GpgA(gamma) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (G betagamma) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway and germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB(alpha)-SfaD(beta)-GpgA(gamma) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination.

Figure 1.

Roles of the G-protein subunits α, β, and γ in trehalose mobilization in response to glucose at the onset of germination. Kinetics of trehalose breakdown is shown in germinating conidia of A. nidulans strains (A) CEA178 (WT), CEA 276 (ganAΔ), CEA278 (ganBΔ), and CEA244 (fadAΔ); (B) CEA209 (WT), CEA 245 (ganBΔ), CEA309 (sfaDΔ), and rJAG19.9 (gpgAΔ); and (C) CEA209 (WT), CEA245 (ganBΔ), CEA293 (rgsAΔ), and CEA294 (rgsAΔganBΔ) inoculated in liquid minimal medium with glucose (1%) at 37°. Trehalose levels in each sample were normalized for the trehalose content in conidia (100%) of the corresponding strain. Results are representative of three (A and B) or two (C) independent experiments.

Figure 2.

Intracellular cAMP levels in germinating conidia of wild-type and ganBΔ strains. Conidia were inoculated in liquid minimal medium at 37° at 2 × 10⁷/ml. After addition of glucose (1%) aliquots of spore suspension were removed at the indicated times and intracellular cAMP levels were measured as indicated in materials and methods. Error bars indicate standard deviations of duplicate samples. Kinetics of cAMP levels are representative of three independent assays.

Figure 3.

The G-proteins GanB (α), SfaD (β), and GpgA (γ) constitute a genetically related complex required for efficient conidial germination. Kinetics of germ tube outgrowth in A. nidulans strains (A) CEA209 (WT), CEA245 (ganBΔ), CEA308 (sfaDΔ), and rJAG19.9 (gpgAΔ) and (B and C) CEA 209 (WT), CEA293 (rgsAΔ), CEA309 (sfaDΔ), and CEA316 (rgsAΔ sfaDΔ) inoculated in liquid minimal medium at 37° in the presence (A and B) or absence (C) of glucose (1%) are shown. The number of conidia showing a germ tube or a protrusion was recorded at different times in at least two microscopic fields and is presented as a percentage of the total number of conidia (100) in these fields. Results are representative of two independent experiments.

Figure 4.

Deletion of cyaA suppresses hypergermination phenotypes caused by deletion of rgsA. Kinetics of (A and B) germ tube outgrowth and (C) trehalose breakdown in germinating conidia of A. nidulans strains (A–C) CEA178 (WT) and CEA179 (cyaAΔ), (A) CEA278 (ganBΔ) and CEA310 (ganBΔ cyaAΔ), and (B and C) CEA312 (rgsAΔ) and CEA314 (rgsAΔ cyaAΔ) inoculated in liquid minimal medium supplemented with glucose (1%) at 37° are shown. The number of conidia showing a germ tube or a protrusion was recorded at different times in at least two microscopic fields and is presented as a percentage of the total number of conidia (100) in these fields. Results of trehalose breakdown and germ tube outgrowth are representative of three independent experiments.

Figure 5.

GanB is required for conidial germination in response to various carbon sources. Conidia of A. nidulans strains CEA209 (WT) and CEA245 (ganBΔ) were germinated at 37° in liquid minimal medium with the indicated carbon sources (1%) and the number of conidia with a germ tube or a protrusion was recorded at 14 hr after addition of the carbon source. Results are representative of three independent experiments. Error bars indicate standard deviations of three independent experiments.

Figure 6.

Proposed model depicting the molecular mechanisms regulating the early events of germination by carbon source sensing in A. nidulans. Sensing of an external carbon source triggers activation of the heterotrimeric G-protein GanB(Gα)-SfaD(Gβ)-GpgA(γ), which in turn initiates the early events of conidial germination through activation the cAMP/PKA pathway. The Gα-subunit (GanB) appears to be the primary signaling element responsible for activation of the cAMP/PKA pathway. The Gβγ-dimer SfaD-GpgA is necessary for proper activation of GanB by carbon source sensing. RgsA is involved in downregulation of cAMP-PKA-dependent germination through inhibition of the GanB activity.