Functional Genomics Approaches In a Model Filamentous Fungus

Neurospora crassa is among the best-characterized model systems for the filamentous fungi. This group of organisms includes many significant animal and plant pathogens, as well as species that are of critical importance to industry. There are 9733 genes in the latest annotation of the Neurospora genome and we have completed the processing of every knockout cassette created from the first pass. Mutants for 8317 genes have been deposited at the Fungal Genetics Stock Center, which distributes the mutants to the scientific community. More than 1200 genes have been analyzed for growth and morphological phenotypes and a subset have also been subjected to chemical sensitivity screens. The group of genes that have been analyzed includes transcription factors, serine-threonine protein kinases, serine-threonine protein phosphatases and genes in other pathways. In a separate study, we are developing a platform for high-throughput assay of protein-protein interactions and co-localization of proteins by engineering strains bearing epitope- and fluorescent protein-tagged open reading frames (ORFs). For this purpose, we constructed two vectors: one that targets V5 and GFP epitope-tagged ORFs to the pan-2 locus, while the other inserts S-tag and RFP epitope-tagged ORFs at the inl locus. Both vectors also carry the bar gene, conferring resistance to Ignite. The targeting vectors disrupt inl or pan-2, yielding pantothenate or inositol auxotrophs, respectively. We are exploiting this auxotrophy to force strains with two different ORFs to fuse (form heterokaryons). This brings the two tagged proteins together in the same cell, allowing possible interactions to be assessed.