Gene discovery and gene function assignment in filamentous fungi.
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.
Hamer
L
L
Paradigm Genetics, 108 Alexander Drive, Research Triangle Park, NC 27709, USA. lhamer@paragen.com
Adachi
K
K
Montenegro-Chamorro
M V
MV
Tanzer
M M
MM
Mahanty
S K
SK
Lo
C
C
Tarpey
R W
RW
Skalchunes
A R
AR
Heiniger
R W
RW
Frank
S A
SA
Darveaux
B A
BA
Lampe
D J
DJ
Slater
T M
TM
Ramamurthy
L
L
DeZwaan
T M
TM
Nelson
G H
GH
Shuster
J R
JR
Woessner
J
J
Hamer
J E
JE
D000483:Alleles; D001203:Ascomycota / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D018556:Crops, Agricultural / Q000382:microbiology; D004251:DNA Transposable Elements / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics* / Q000502:physiology; D015698:Genomic Library; D020461:Madurella / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010641:Phenotype; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D005656:Fungal Proteins