Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora

Abstract

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

Figure 1.—

Comparison of amino acid sequences from S. macrospora (Sm) GSA1, GSA2, and GSA3 proteins (accession nos. CAP09209, CAP09210, and CAP09211) with their N. crassa (Nc) orthologs (accession nos. AAB37244.1, Q05424, and XP_962205.1). The amino acids with the solid background are identical in all three subunits of both species. Sequence identity between four or five sequences is indicated by shading. Conserved regions that are predicted to play a role in the interaction with GTP are underlined. Putative myristoylation sites are marked with boxes. Amino acid identity of Gα-subunits is given at the end in percentages.

Figure 2.—

Fruiting body development and ascospore germination of wild type, Δgsa, and Δsac1 mutant strains. r2 and fus1 are spore color mutants that show wild-type fertility. (A) Perithecia on solid cornmeal medium after 11 days of growth. The scale bar represents 1 mm. (B) Ascospore germination of wild-type and mutant strains Δsac1 and Δgsa3/r2. Spores were incubated for 5 hr on solid cornmeal medium with 0.5% (w/v) sodium acetate. The scale bar represents 50 μm. (C) Lateral view on perithecia of wild type, Δsac1, Δsac1/fus1 supplemented with cyclic AMP (+cAMP), and retransformant Δsac1 + cr-1 on solid cornmeal medium after 9 days of growth. The scale bar represents 200 μm.

Figure 3.—

Phenotypic characterization of fruiting body development of Gα-single and -double mutants. (A) Perithecial development on solid cornmeal medium after 11 days. Bar, 1 mm. (B) Microscopic images of protoperithecia from wild-type and double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3/r2. Bar, 10 μm.

Figure 4.—

Phenotypic characterization of fruiting body development in Gα-subunits/pheromone receptor mutants. Δpre receptor mutants (A) and ΔgsaΔpre double mutants (B) are shown after growth for 11 days on solid cornmeal medium. (C) Transformation with gsa1 (+gsa1) restores fertility in the Δgsa1Δpre mutants. Bar, 1 mm.

Figure 5.—

Phenotypic characterization of Δgsa/Δsac1 double mutants and Δgsa2Δgsa3Δsac1 triple mutant. (A) Perithecial development of wild type, Δsac1, and Δgsa/Δsac1 double mutants after 11 days of growth on solid cornmeal medium. Bar, 1 mm. (B) Protoperithecial development in the sterile double mutant Δgsa1/Δsac1. Bar, 10 μm. (C) Δgsa2Δgsa3Δsac1 triple mutant after 11 days of growth on solid cornmeal medium.

Figure 6.—

Phenotypic characterization of Δste12 single and ΔgsaΔste12 double mutants. (A) Perithecial development of Δste12 single and Δgsa/Δste12 double mutants after 11 days of growth on solid cornmeal medium. Bar, 1 mm. (B) Microscopic images of asci from Δste12 single and Δgsa1Δste12 and Δgsa2Δste12 double mutants after 11 days of growth on solid cornmeal medium. The Δgsa3Δste12 mutant does not produce perithecia and therefore lacks any asci. Bar, 100 μm.

Figure 7.—

Comparison of transcript levels of pro41 and ste12 between different mutants and phenotypes of pro41Δgsa double mutants. (A) Quantitative real-time PCR data are given as logarithmic values of the mutant/wild-type ratios (logarithm to the base 2 for the mean of at least two independent experiments). Real-time PCR results were tested for the significance of differential expression at P = 0.001 using REST (Pfaffl et al. 2002); genes that are expressed significantly differently in the mutant compared to the wild type are indicated by an asterisk. (B) Perithecial development of pro41 single and pro41Δgsa double mutants after 11 days of growth on solid cornmeal medium. Bar, 1 mm.

Figure 8.—

A model for the predicted G protein α-subunit signaling in S. macrospora. GSA1 and GSA2 propagate signals within the pheromone signaling pathway, in which GSA1 is the predominant regulator of fruiting body development upstream of the STE12 transcription factor. GSA3 and SAC1 act on sexual development in a less characterized, parallel signaling pathway. Putative myristoylation of Gα-subunits GSA1 and GSA3 is indicated by tails. Putative farnesylation of PPG2 is shown by a serrated tail.