Katherine A. Borkovich

We have identified a new gene encoding the G protein α subunit,gna-3, from the filamentous fungusNeurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Gα 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 Δgna-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 Δgna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Δgna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Δgna-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 Δgna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Δgna-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.

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1 , from the eukaryotic filamentous fungus Neurospora crassa . The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Δ nop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all- trans [ ³ H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.

Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gα_i superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1^R178C and gna-1^Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1^R178C and gna-1^Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1^R178C and gna-1^Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1^R178C and gna-1^Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

Heterotrimeric G proteins, consisting of α, β and γ subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein α subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Δgna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a Δgna-1 strain accentuates the Δgna-1 phenotypes. Δgna-1 Δgna-2 strains have a slower rate of hyphal apical extension than Δgna-1 strains on hyperosmotic media. Moreover, Δgna-1 Δgna-2 mutants have more pronounced defects in female fertility than Δgna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein α subunits with overlapping functions in eukaryotic microbes.