Filamentous fungi such as Neurospora crassa use G protein coupled receptors (GPCRs) and associated heterotrimeric G proteins to respond to sensory cues from the environment. A fundamental challenge in G protein signaling is to identify the specific subunits of the heterotrimer (Gα, Gβ, Gγ) that interact in cases where multiple versions of subunits are present. Relevant to downstream signaling by heterotrimeric G proteins, fungi differentially regulate secreted enzymes for catabolism of cellulose, but the connection between cellulose metabolism and any signal transduction pathway is lacking in N. crassa. The primary objectives of this thesis are to 1. Determine genetic relationships between the putative Gβ subunit CPC-2 and the three Gα protein subunits in N. crassa, 2. Investigate the extent of G protein involvement in the cellulose response in N. crassa, and 3. Determine the role of N. crassa G proteins on the global transcriptional response to cellulose as a carbon source. In Chapter 2, we characterized the relationships between the Gβ subunit CPC-2 and the other known G protein subunits in N. crassa on minimal medium containing sucrose. We illustrated that CPC-2 is cytoplasmic. We also demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height. Strains lacking both Gβ subunits possessed more severe defects for all phenotypic traits except for production of macroconidia, supporting a synergistic relationship between GNB-1 and CPC-2 in N. crassa.
In Chapter 3, I examined the relationship that G protein signaling has with cellulose metabolism. Loss of the Gα subunits gna-1 and gna-3, the Gβ subunits gnb-1 and cpc-2, the Gγ gng-1, or adenylyl cyclase (cr-1) resulted in loss of detectable cellulase activity. The expression patterns for five cellulase genes revealed that Δgna-1, Δgnb-1, and Δgna-3 mutants produce less cellulase mRNA than wild type, consistent with transcriptional regulation. Δcpc-2 and Δcr-1 mutants had wild-type levels of the cellulase transcripts. These results suggest that CPC-2 and CR-1 affect cellulase production in a post-transcriptional manner. Moreover, cAMP addition only partially corrected cellulase activity defects in Δgna-1 and Δgnb-1 mutants, indicating that GNA-1 and GNB-1 target cAMP-independent pathways to control cellulase activity.
In Chapter 4, I analyzed the transcriptomes and exoproteomes from cellulose grown cultures of the mutants for the Gα subunits gna-1 and gna-3, as well as the adenylate cyclase cr-1 via RNAseq and LC/MS-MS protein identification. 20 of the 22 highly expressed cellulases found in wild type cultures were transcriptionally downregulated in Δgna-1 mutants, and 6 of these 20 were also down-regulated in Δgna-3 mutants. Δcr-1 mutants were not transcriptionally downregulated for any cellulase enzymes. Our transcriptional data suggests that gna-1 and gna-3 control the response to cellulose in N. crassa, while cr-1 affects cellulases in a post-transcriptional manner.
