Abstract
Heterotrimeric (αβγ) G protein signaling pathways are critical environmental sensing systems found in eukaryotic cells. Exchange of GDP for GTP on the Gα subunit leads to its activation. In contrast, GTP hydrolysis on the Gα is accelerated by Regulator of G protein Signaling (RGS) proteins, resulting in a return to the GDP-bound, inactive state. Here, we analyzed growth, development and extracellular cellulase production in strains with knockout mutations in the seven identified RGS genes (rgs-1 to rgs-7) in the filamentous fungus, Neurospora crassa. We compared phenotypes to those of strains with either knockout mutations or expressing predicted constitutively activated, GTPase-deficient alleles for each of the three Gα subunit genes (gna-1(Q204L), gna-2(Q205L) or gna-3(Q208L)). Our data revealed that six RGS mutants have taller aerial hyphae than wild type and all seven mutants exhibit reduced asexual sporulation, phenotypes shared with strains expressing the gna-1(Q204L) or gna-3(Q208L) allele. In contrast, Δrgs-1 and Δrgs-3 were the only RGS mutants with a slower growth rate phenotype, a defect in common with gna-1(Q204L) strains. With respect to female sexual development, Δrgs-1 possessed defects most similar to gna-3(Q208L) strains, while those of Δrgs-2 mutants resembled strains expressing the gna-1(Q204L) allele. Finally, we observed that four of the seven RGS mutants had significantly different extracellular cellulase levels relative to wild type. Of interest, the Δrgs-2 mutant had no detectable activity, similar to the gna-3(Q208L) strain. In contrast, the Δrgs-1 and Δrgs-4 mutants and gna-1(Q204L) and gna-2(Q205L) strains exhibited significantly higher cellulase activity than wild type. With the exception of sexual development, our results demonstrate the greatest number of genetic interactions between rgs-1 and gna-1 and rgs-2 and gna-3 in N. crassa.