A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

Abstract

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.

Fig. 1.

Strategy for creating deletion constructs. 5′ and 3′ flank fragments are amplified separately from genomic DNA with primers 5f + 5r and 3f + 3r. Primers 5r and 3f incorporate MmeI sites (M) and have 5′ tails homologous to the hph cassette, whereas those for 5f and 3r are homologous to the vector. The two flanks are cotransformed into yeast along with the hph cassette and gapped yeast shuttle vector. Homologous recombination creates the circular construct and the final linear deletion cassette is amplified from the pooled yeast DNA with primers 5f and 3r. hph is transcribed in the antisense direction relative to the target gene.

Fig. 2.

Venn diagram showing the distribution of transcription factor knockout mutants with observed phenotypes. Mutants are represented by the NCU numbers of the deleted genes. Font color indicates gene family (see Inset table). Miscellaneous genes (pink) are from three classes: RING-type zinc finger (NCU06411), CBF CAAT-binding factor (NCU02017), and homeobox (NCU00097 and NCU03593). Genes showing ascospore lethality are shown in the oval. The numbers of transcription factor mutants analyzed and the numbers with phenotypes are listed in the Inset table. Knockout mutants indicated with * or + exhibited greater basal hyphal extension rates or aerial hyphae heights than wild type, respectively. For the wild-type controls, the basal hyphae extension rate was 65–80 mm per day, whereas the height of aerial hyphae achieved in 3 days was 30–45 mm. The three photographs show a perithecium (Upper), a conidiophore (Lower Right), and basal hyphae (Lower Left).

Fig. 3.

Colony morphology and asexual and sexual development of transcription factor knockout mutants. (A) Colony morphology of wild type and Δkal-1:NCU03593. Strains were grown for 24 h on VM or VM + yeast extract at 25°C and 37°C. The colony edge images show basal vegetative hyphae at ×11. (B) Transcription factor mutant exhibiting aberrant development of perithecia. Images at ×77 were taken 7 days after fertilization of protoperithecia with opposite mating type conidia. The arrow indicates a beak in wild type; this structure does not form in the mutant.