Elucidation of G-Protein Signal Transduction in Neurospora crassa Using Chemical Genomics, Metabonomics, and Genetics

The filamentous fungus Neurospora crassa is a model organism for filamentous fungi and has been studied for many years due to its many advantages as an organism for study. Conidiation is a major mode of dispersal utilized by fungal pathogens, and it is evident that conidiation is a common biological response to adverse conditions and a means by which the fungus can reestablish itself in a more favorable environment. N. crassa produces a type of asexual spore, the macroconidium (referred to as a conidium) for dissemination in the environment. In order to interact with its surroundings, fungi must be able to receive extracellular signals. A major signaling pathway that detects and responds to external signals in fungi and other eukaryotes is mediated by heterotrimeric GTP-binding proteins (consisting of alpha, beta, and gamma subunits). Three G alphas (GNA-1, GNA-2, and GNA-3), one G beta (GNB-1) and one G gamma subunit (GNG-1) have been identified in N. crassa. Loss of gna-3 has dramatic effects on conidiation, leading to production of short aerial hyphae and premature conidiation in plate cultures and inappropriate conidiation in submerged cultures. RIC8 is a novel guanine exchange factor (GEF) found in Neurospora crassa and has been shown to also interact with GNA-1 and GNA-3 in N. crassa. Yeast2Hybrid cDNA library screening has shown that RIC8 interacts with STE50, an adaptor protein working in the pathway of MAPK. A ric8 deletion mutant shares several phenotypes with ste50 deletion mutants. Also, without STE50, CAT formation is compromised. Localization of STE50 was investigated using a GFP-fused STE50; it localized to the cytoplasm as well as to the hyphal septa. Finally, coimmunoprecipitation (Co-IP) to verify that RIC8 and STE50 do physically interact was carried out. As a participating student of the IGERT (Integrative Graduate Education and Research Traineeship), I was privileged to partake in a chemical genomics study; my goal was to find a chemical that perturbed asexual sporulation (conidiation) or spore germination in Neurospora crassa. From screens of the Chembridge library, I identified a compound that inhibited conidiation of gna-3 deletion mutants. However, subsequent testing showed that newer batches of the chemical did not elicit the same phenotype; hence, this compound was dropped. However, during the screening of the Chembridge library, a unique phenotype was observed from one of the compounds that was tested. Compound #5979758 caused a "blebbing" effect on the conidia of all Neurospora strains tested so far. Furthermore, I entered into a collaboration with Dr. Cythia Larive and Kayla Kaiser to use proton NMR to study metabonomics in gna-3 deletion mutant and wild-type strains under different conditions. The metabolic profiles for wild type and gna-3 deletion mutants were well-documented. From this study, it was revealed that the metabolome of gna-3 deletion mutant does not vary greatly from that of the wild-type, even though it conidiates in submerged conditions. Also, it was shown that GNA-3 is involved in a nutrient sensing pathway. A paper titled "Use of proton NMR to measure intracellular metabolite levels during growth and asexual sporulation in Neurospora crassa", with authors James Kim, Kayla Kaiser, Cynthia Larive and Katherine Borkovich, was published in the journal Eukaryotic Cell