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The Role of Heterotrimeric G-Proteins, Regulators of G-Protein Signaling, and Adenylyl Cyclase in Regulating Cellulase Expression and Activity in the Model Filamentous Fungus Neurospora crassa
Previous work from our group reveals that filamentous fungi utilize G-protein signaling for cellulose sensing and degradation. The primary objectives of this dissertation are: 1. to determine the role of Regulator of G-protein Signaling (RGS) proteins in the regulation of Gα subunits during growth on cellulose, 2. to elucidate global transcriptional regulation by G-protein subunits and their downstream effectors on cellulose, and 3. to examine regulation of specific cellulase enzyme families by G-proteins, RGS proteins, and adenylyl cyclase.In Chapter 2, we characterized the role of RGS proteins in growth, development, and cellulase activity in N. crassa. Through direct inoculation into Avicel (cellulose), I showed that RGS-2 is essential for Avicelase activity. I also found RGS-1 and RGS-4 as negative, and RGS-3 as positive regulator of Avicelase activity. RGS-1 negatively regulated culture biomass on Avicel while RGS-2, RGS-3, and RGS-7 were positive regulators.
Chapter 3 involved analysis of global transcriptomes of Δgna-1, Δgna-3, and Δcr-1 mutants grown on glucose and cellulose. Upon comparing the transcriptomes of mutants to wild-type strain, I found that GNA-3 regulates the greatest number of genes on glucose, while the adenylyl cyclase CR-1 regulates the most genes on cellulose. I identified that over-expression of CLR-2, a transcriptional regulator of cellulases, rescued Avicelase activity in Δgna-1 and Δgna-3 mutants to nearly wild-type levels. I also acquired evidence about the role of CLR-2 in deregulation of macro-conidiation in submerged cultures through VIB-1.
In Chapter 4, I performed specific cellulase assays for endoglucanases (EG), cellobiohydrolases (CBH), and β-glucosidases (BG) on G-protein single and multiple gene deletion mutants, RGS mutants, the cr-1 mutant, and GTPase-deficient Gα strains. Major results show that CR-1 regulates CBH activity through GNA-2 and GNA-3. I also show that GNA-1 and GNA-3 together regulate EG activity. RGS proteins are observed to regulate cellulase enzyme activities to a moderate level. The GTPase-deficient gna-1 allele showed elevated EG and CBH activities. These results show that G-protein signaling contributes more to EG and CBH activities than to BG activity.
Overall, this dissertation identifies the role of RGS proteins in cellulose degradation and elucidates the regulation of cellulase activity by G-proteins in further depth
Regulator of G Protein Signaling Proteins Control Growth, Development and Cellulase Production in Neurospora crassa
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-1Q204L, gna-2Q205L or gna-3Q208L). 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-1Q204L or gna-3Q208L 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-1Q204L strains. With respect to female sexual development, Δrgs-1 possessed defects most similar to gna-3Q208L strains, while those of Δrgs-2 mutants resembled strains expressing the gna-1Q204L 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-3Q208L strain. In contrast, the Δrgs-1 and Δrgs-4 mutants and gna-1Q204L and gna-2Q205L 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