Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature‐sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature‐sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

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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TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.