Tools for Functional Analysis and Cell Biology

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The central goal of the three interdependent efforts in this Program Project builds upon successful functional genomics, annotation, and expression analyses of Neurospora crassa, a premier filamentous fungus model for over 250,000 species of non-yeast fungi. A primary goal, targeted through molecular, cell biological, genomic and computational approaches, will be to understand how N. crassa transitions from mycelial growth to complete asexual spore development. We will focus on two key triggers of asexual development: light and desiccation. In addition, we will leverage our prior successes to expand systematic knockouts to an additional prominent model system, Aspergillus nidulans. Neurospora is a salient model for basic research in eukaryotes, as is Aspergillus, and fungi allied to these species include most animal and plant pathogens as well as industrial strains yielding antibiotics, chemicals, enzymes, and Pharmaceuticals. Gene predictions among the 9846 genes encoded by the fully sequenced 43 Mb Neurospora genome are supported by over 250,000 ESTs derived from this P01. During the past 4 years, we revolutionized the tools and techniques for gene knockouts in filamentous fungi and exceeded our initial goal by over 40%. Project #1 will complete the Neurospora gene knockouts and extend the systematic disruption of genes to Aspergillus. We will develop strains, tools, and background information in support of Projects #2 and #3, and of the overarching goal of understanding regulatory pathways governing filamentous fungal development. Projects #2 and #3 will aim to describe and reconstruct the cascading regulatory programs that underlie N. crassa's developmental response to light and air, from the level of chromatin structure through the gene regulatory network. Project #3 will use ChlP-seq mapping of histone modifications, transcription factor binding sites, sites of DNA methylation and nucleosome occupancy with corresponding transcriptome measurements to generate a deep description of genome and epigenome dynamics. Project #2, in an informatic-intensive systems biology approach, will integrate these data and use computational modeling to develop predictive models of the interconnected light and asexual development gene regulatory networks. These models will be fleshed out tested through incorporation of new data arising from Project #3 and refined via perturbation analyses facilitated through the use of strains developed in Project #1 and characterized using the tools employed in Project #3. This effort will anchor, promote, and exploit genomic exploration within model systems that provide gateways to the Kingdom of the Fungi.