Gene discovery and gene function assignment in filamentous fungi

doi:10.1073/pnas.091094198

Gene discovery and gene function assignment in filamentous fungi

^*Paradigm Genetics, 108 Alexander Drive, Research Triangle Park, NC 27709; and ^‡Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282

Communicated by R. James Cook, Washington State University, Pullman, WA (received for review January 9, 2001)

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 transposon-arrayed 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.

Footnotes

↵ † To whom reprint requests should be addressed at: Paradigm Genetics, 108 Alexander Drive, Building 1A, Research Triangle Park, NC 27709. E-mail: lhamer{at}paragen.com.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession no. AF32553 (M. grisea HPD4)].
Abbreviations:

TAGKO,

transposon-arrayed gene knockout;

TI,

targeted integration;

IVT,

in vitro transposition;

IM,

integration mutant;

WT,

wild type;

MM,

minimal medium;

MAC1,

Magnaporthe grisea adenylate cyclase gene;

HPD4,

p-hydroxyphenylpyruvate dioxygenase