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

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 gsagenes (Delta gsa1, Delta gsa2, and Delta gsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for G alpha-subunits have distinct roles in the sexual life cycle. While single Mutants show some reduction of fertility, double mutants Delta gsa1 Delta gsa2 and Delta gsa1 Delta gsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct G alpha-subunits, two recently generated Delta pre strains were crossed with all Delta gsa 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) (sac]) for construction of a knockout strain. Analyses of the three Delta gsa Delta sac1 double mutants and one Delta gsa2 Delta gsa3 Delta sac1 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 SLIM of all our data, we propose a model for how different G alpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.