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I received my B.S. in biochemistry from the University of California, Davis, in 1979. I went on to obtain my Ph.D. in biochemistry in 1985 at the University of California, Los Angeles. My thesis research encompassed characterization of the kinetics, associated metal cofactor and expression of the enzyme arginase from the filamentous fungus Neurospora crassa (Figure 1). My postdoctoral work in Susan Lindquist's laboratory at the University of Chicago was focused on the functions of the heat-shock protein genes HSP83/HSC83 and HSP104 from the yeast Saccharomyces cerevisiae. I determined that HSP83/HSC83 comprises an essential gene family, with higher concentrations of these proteins required for growth at higher temperatures. I then joined Melvin Simon's laboratory at the California Institute of Technology as a postdoctoral fellow. There, I investigated coupling of transmembrane chemoreceptors to intracellular histidine kinase activity during regulation of chemotaxis in the bacterium Escherichia coli. I demonstrated ligand-dependent transfer of phosphoryl groups in vitro, using both attractant and repellent stimuli. I also explored the effect of receptor adaptation on histidine kinase activity. I became an assistant professor in the Department of Microbiology and Molecular Genetics at the University of Texas-Houston in 1991. My group began study of heterotrimeric G protein genes from filamentous fungi, using the model organism Neurospora crassa. Subsequently, we initiated a project to investigate a possible role for opsins in the photobiology of Neurospora. I transferred my laboratory to the University of California, Riverside, in 2001. I was promoted to Associate Professor with tenure at UCR in July 2003 and to Full Professor in July 2005. Since 2004 my laboratory has received funding from a NIH Program Project to design and implement a high-throughput gene knockout procedure for Neurospora. We have recently begun analysis of Neurospora two-component regulatory systems and are also applying chemical genetics screens to study of G protein signaling in this organism.
Heterotrimeric G Proteins in Filamentous Fungi In eukaryotic organisms, major signal transduction pathways utilize heterotrimeric (αβγ) G proteins. G proteins are involved in numerous processes in mammals, including vision, taste, smell, cardiac function and neurotransmission. G protein activation involves ligand binding by a seven transmembrane helix G protein-coupled receptor (GPCR), GDP/GTP exchange on the Gα subunit and dissociation of the heterotrimer, freeing Gα and Gβγ to regulate downstream effectors. My research resulted in the discovery of heterotrimeric G proteins in filamentous fungi, using the model organism Neurospora crassa. This field has opened up several new areas, including study of G protein-mediated virulence in plant and animal pathogens. Another breakthrough was our finding that filamentous fungi contain a Gα subunit belonging to a superfamily found in higher organisms. This result is supported by evolutionary studies showing that filamentous fungi are more closely related to mammals than are yeasts. My laboratory has characterized the three Gα (GNA-1, GNA-2 and GNA-3), one Gβ (GNB-1) and one Gγ (GNG-1) proteins in Neurospora. We have demonstrated that G proteins are critical for proliferative growth and differentiation in filamentous fungi, controlling growth rate, asexual sporulation, female fertility and stress tolerance. We obtained the first evidence for Gα subunits with overlapping functions in eukaryotic microbes. We have shown that adenylyl cyclase activity is regulated by two different mechanisms involving Gα proteins; this is significant, because adenylyl cyclase is also regulated by Gα proteins in mammals. This finding was featured on the cover of Molecular and Cellular Biology in 2000. We have demonstrated that G proteins regulate both cAMP dependent and independent functions in Neurospora. We have shown that loss of the Gβ or Gγ subunit leads to lower levels of Gα proteins under a variety of conditions, a trait shared with at least one other filamentous fungus and also suggested by experiments in mammalian systems. We have identified 10 GPCRs in the Neurospora genome sequence, including three proteins that are similar to predicted GPCRs in slime molds, Arabidopsis and Caenorhabditis elegans, but not found in yeasts. Mutational analysis indicates that pheromone GPCRs regulate pheromone-dependent chemotropic growth of female hyphae towards male cells, while at least one member of the novel GPCR class is required for maturation of fertilized reproductive structures (Figure 2). Another GPCR is required for growth and development on poor carbon sources such as glycerol. Analysis of the remaining GPCRs and the downstream effector pathways is underway.
Since our original discovery, other laboratories have shown that GNA-1 or GNA-3 homologues are required for pathogenesis in filamentous fungi, including the human pathogen Cryptococcus neoformans and the plant pathogens Aspergillus nidulans, Ustilago maydis, Magnaporthe grisea and Cryphonectria parasitica. We have demonstrated that there are common regulatory mechanisms for regulation of growth and development in Neurospora and pathogenic species. Knowledge of the molecular details of early events in pathogenesis will allow development of new control practices and/or chemical agents to combat fungal diseases in both plants and animals.
Selected Publications Related to Heterotrimeric G Proteins in Filamentous Fungi (Bibliography page) Genome-Wide Analysis of Neurospora As part of a Neurospora community annotation project (Broad Institute, M.I.T.), I coordinated a large group of scientists focused on genes required for growth and development. The analysis resulted in a 2003 paper in Nature describing the sequencing and initial annotation of the Neurospora genome sequence. More recently, I was the first and corresponding author for a Neurospora genome review that was published in Microbiology and Molecular Biology Reviews in 2004. Production of this 108-page paper required that I organize a large number of scientists to annotate 1100 Neurospora genes. I am Co-PI of a NIH Program Project grant for functional genomics, transcriptional profiling, EST sequencing and manual annotation of the Neurospora genome that was awarded in 2004. My laboratory is involved in the genome-wide targeted gene replacement project, with the goal of mutating all of the more than 10,000 ORFs in five years (Figure 3). During the first two years we have created the recipient strains, significantly improved the mutation protocol and established a laboratory information management system (LIMS) for tracking the samples. We created hundreds of mutants that have been sent to a central stock center for distribution to the scientific community. A manuscript summarizing our analysis of 103 transcription factor mutants is in press at the Proc. Natl. Acad Sci., USA.
Selected Publications Related to Genome-Wide Analysis of Neurospora (Bibliography page)
Two-Component Regulatory Systems Two-component regulatory systems, consisting of proteins containing histidine kinase and/or response regulator domains, regulate signal transduction in bacteria, slime molds, fungi and plants. These cascades regulate a diverse array of functions, ranging from responses to nutritional, stress or chemical signals to multicellular development, chemotaxis and light sensing. The genome sequence of Neurospora predicts 11 hybrid histidine kinase, one histidine phosphotransfer protein (HPT) and two response regulator genes. The number of histidine kinase genes is striking, in that the sequenced yeast genomes contain only 1-3 histidine kinase genes. My laboratory has mutated one of the response regulator genes and has submitted a manuscript summarizing our results. We showed that one two-component system is required for osmotic and fungicide resistance and female fertility, through phosphorylation of a downstream Mitogen-Activated Protein Kinase signaling pathway (Figure 2). We have also mutated the hybrid histidine kinases and phenotypic analysis of these strains is underway.
I am a participant in the new NSF ChemGen IGERT training grant here at UCR that is focused on screens to identify chemicals that affect the biology of plants and plant pathogens. Mr. James Kim, an IGERT student, has joined my group and is currently screening the ChemBridge chemical library (10,000 chemicals). He is looking for effects on 1) spore germination and 2) inappropriate asexual spore formation in G protein mutants using Neurospora as a model system for related plant and animal pathogens. These screens are extremely relevant, as asexual spores are the primary means by which pathogenic fungi are disseminated in the environment. Furthermore, strains lacking the Gα subunits gna-1 and gna-3 exhibit a profound growth defect, and some of our G protein mutants sporulate inappropriately in liquid culture. Thus, we can use our existing mutants to determine patterns of sensitivity to particular chemicals and gain clues to the overall mechanism of their action. Identification of chemicals that block spore formation and/or germination would provide candidates for fungicides and antifungal drugs. Mr. Kim has already identified two chemicals that inhibit germination and another that blocks sporulation and he is currently performing experiments to better understand their mode of action. We are very excited about these results, as this is the first application of chemical screens to Neurospora and it gives us a new tool to study G protein signaling in this system.
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