Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important m icroorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We deve loped transposon-arrayed gene knockouts (TAGKO) to discover genes and simul taneously create gene disruption cassettes for subsequent transformation an d mutant analysis. Transposons carrying a bacterial and fungal drug resista nce marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon in sertion sites, and cosmid inserts are liberated to direct insertional mutag enesis events in the genome. Based on saturation analysis of a cosmid inser t and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to in vestigate an amino acid oxidation pathway in two important fungal phytopath ogens.