Regulation of Conidiation and Adenylyl Cyclase Levels by the Gα Protein GNA-3 in Neurospora crassa

Abstract

We have identified a new gene encoding the G protein ␣ subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the G␣ proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the ⌬gna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to ⌬gna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submergedculture conidiation is refractory to cAMP but is suppressed by peptone. In addition, ⌬gna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. ⌬gna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of ⌬gna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the ⌬gna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.G-protein-coupled receptors (GPCRs) are a family of seven transmembrane helix receptors that bind ligands such as neurotransmitters, pheromones, and odorants. GPCRs are associated with heterotrimeric G proteins, consisting of ␣, ␤, and ␥ subunits (for a review, see reference 82). In the inactive state, the heterotrimer is docked at the receptor and GDP is bound to the G␣ subunit. Binding of a ligand activates the receptor, leading to the exchange of GDP for GTP on G␣ and the subsequent dissociation of G␣-GTP from the G␤␥ moiety. G␣-GTP and the G␤␥ dimer can activate downstream effectors, such as enzymes and ion channels, to produce a response to the signal (for a review, see reference 7). The response is terminated and the cycle is completed with hydrolysis of GTP by G␣. The GDP-bound G␣ protein reassociates with G␤␥, and the heterotrimer is then able to bind to the receptor to await the next cycle of activation (for reviews, see references 10 and 20).Neurospora crassa is a filamentous fungus that has a complex life cycle due to its ability to produce both asexual and sexual spores (for a review, see reference 71). Grown with adequate nitrogen, N. crassa remains in the asexual cycle and extends basal hyphae to form a complex, intertwined network called a mycelium. Asexual spores, or conidia, are produced by two pathways: macroconidiation and micr...

Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

“…This finding is similar to the DGPA2 mutant of S. pombe and to the defect observed in the corresponding Dgna-3 mutant from N. crassa, indicating that activation of this pathway might be an essential step in the germination process (Kays et al 2000;Hatanaka and Shimoda 2001). Interestingly, the corresponding Dgna-3 mutant from N. crassa develops smaller perithecia that are submersed in the agar (Kays et al 2000), a phenotype that was not observed in the corresponding S. macrospora mutant, but in the Dsac1 disruption strain. The differences between Dgsa1, Dgsa2, and Dgsa3 mutants suggest that they are involved in different steps of sexual development, such as fruiting body development and germination of the ascospores.…”

“…These two features indicate that GSA1, just like its N. crassa counterpart, is evolutionarily related to the Ga i -subfamily of mammals that inhibits adenylyl cyclase (Ivey et al 1996). As already shown by others, the amino acid sequences for Ga-subunits are highly homologous within filamentous fungi (Bö lker 1998; Kays et al 2000;Parsley et al 2003). The proteins encoded by gsa1, gsa2, and gsa3 from S. macrospora display significant identities to corresponding proteins from other fungi with the highest amino acid identity to the Ga-subunits from N. crassa (data not shown).…”

“…The third fungal Ga-subunit placed in group II has no known mammalian counterpart (Bölker 1998). Currently, the best studied example among filamentous fungi is Neurospora crassa, where the three Ga-subunits are known to contribute significantly to sexual and vegetative development (Ivey et al 1996;Baasiri et al 1997;Kays et al 2000;Kays and Borkovich 2004). In this heterothallic fungus, disruption of the genes encoding the Ga-subunits has an effect not only on fruiting body development, but also on the fertilization process.…”

Trichoderma: sensing the environment for survival and dispersal

“…The Ga proteins can be classified into three major subgroups: I, which inhibit adenylate cyclase; II, which have no homology with mammalian G proteins; and III, which in most fungi stimulate adenylate cyclase. In filamentous fungi, heterotrimeric G protein signalling pathways are involved in sporulation, mating, pathogenicity, secondary metabolite production, and vegetative incompatibility (Kays et al, 2000;Rosén et al, 1999;Horwitz et al, 1999;Loubradou et al, 1999).…”

Roles for Receptors, Pheromones, G Proteins, and Mating Type Genes During Sexual Reproduction in Neurospora crassa

“…Growth rate, hyperosmotic sensitivity, conidiation, heterokaryon formation, and CAT fusion assays: Apical extension rates, hyperosmotic sensitivity, morphology of submerged cultures, and growth in standing liquid cultures were examined as previously described (Ivey et al 1996;Kays et al 2000;Kim and Borkovich 2004). Formation and fusion of conidial anastomosis tubes were assayed (Fleissner et al 2005;Kim and Borkovich 2006).…”