Gene discovery and gene function assignment in filamentous fungi

Abstract

Filamentous fungi are a large group of diverse and economically important microorganisms. 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 developed transposonarrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert 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 investigate an amino acid oxidation pathway in two important fungal phytopathogens.A powerful asset for functional genomic analysis is the ability to create large annotated single gene mutant collections. For model research organisms such as baker's yeast, Drosophila, Caenorhabditis, Arabidopsis, and mice, whole genome knockout collections (1), transposon lines (2), or insertional mutant collections (3, 4) are well developed. However, in addition to these model organisms there is a vast array of economically important organisms where genome sequences and functional genomic technologies are lacking. For example, filamentous fungi are causal agents of severe human (5, 6) and crop (7, 8) diseases, and many others are being exploited in the fermentation and food industries (9). Few of these fungal genomes have been analyzed, and large-scale approaches to functional analysis are needed.The model fungus, Saccharomyces cerevisiae, has Ϸ6000 genes in 12 Mb of DNA sequence (10). Targeted integration (TI) for creating gene-specific mutations is very efficient and requires only 50-bp fragments of target gene homology on either side of a selectable marker (11,12). In contrast, many filamentous fungi have genome sizes in the range of 30-40 Mb and are estimated to contain at least 10,000 genes (13). Genome studies using expressed sequence tag analysis suggest that more than half of these genes lack homologues in S. cerevisiae (14). TI occurs at very low frequencies (1-20%) for many filamentous fungi, and larger fragments of target gene homology must be used to obtain targeted insertion events (15).To initiate genome-wide mutagenesis studies in filamentous fungi we developed an approach we call transposon-arrayed gene knockouts (TAGKO) (Fig. 1). In vitro transposition (IVT) (16-19) into cosmid libraries is used to create gene sequencing templates. Subsequent sequencing and analysis from these templates creates an annotated collection of insertional gene disruption vectors. We demonstrate that IVT can...

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