A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.
The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
Colot
Hildur V
HV
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755.
Park
Gyungsoon
G
Department of Plant Pathology, University of California, Riverside, CA 92521; and.
Turner
Gloria E
GE
Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095.
Ringelberg
Carol
C
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755.
Crew
Christopher M
CM
Department of Plant Pathology, University of California, Riverside, CA 92521; and.
Litvinkova
Liubov
L
Department of Plant Pathology, University of California, Riverside, CA 92521; and.
Weiss
Richard L
RL
Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095.
Borkovich
Katherine A
KA
Department of Plant Pathology, University of California, Riverside, CA 92521; and.
Dunlap
Jay C
JC
*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755; jay.c.dunlap@dartmouth.edu.
D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D025301:Hyphae; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009491:Neurospora / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010641:Phenotype; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005656:Fungal Proteins; D014157:Transcription Factors