Regulator of G Protein Signaling Proteins Control Growth, Development and Cellulase Production in Neurospora crassa

Heterotrimeric (alpha beta gamma) G protein signaling pathways are critical environmental sensing systems found in eukaryotic cells. Exchange of GDP for GTP on the G alpha subunit leads to its activation. In contrast, GTP hydrolysis on the G alpha 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 G alpha 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, Delta rgs-1 and Delta rgs-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, Delta rgs-1 possessed defects most similar to gna-3(Q208L) strains, while those of Delta rgs-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 Delta rgs-2 mutant had no detectable activity, similar to the gna-3(Q208L) strain. In contrast, the Delta rgs-1 and Delta rgs-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.