Fungal functional genomics: tunable knockout-knock-in expression and tagging strategies

Abstract

Strategies for promoting high-efficiency homologous gene replacement have been developed and adopted for many filamentous fungal species. The next generation of analysis requires the ability to manipulate gene expression and to tag genes expressed from their endogenous loci. Here we present a suite of molecular tools that provide versatile solutions for fungal high-throughput functional genomics studies based on locus-specific modification of any target gene. Additionally, case studies illustrate caveats to presumed overexpression constructs. A tunable expression system and different tagging strategies can provide valuable phenotypic information for uncharacterized genes and facilitate the analysis of essential loci, an emerging problem in systematic deletion studies of haploid organisms.

FIG. 1.

Locus-specific tunable expression and N- and C-terminal tagging strategies. (A) General strategy for N- and C-terminal modification of fungal genes using cassettes generated by YRC and PCR. The left panel shows the replacement of an endogenous promoter by the inducible qa-2 promoter, with the option of adding a V5 (or other) epitope to the gene product of interest. The right panel depicts the addition of a tag (V5 epitope six-His) at the C terminal by the same homologous recombination-based strategy. (B) The QA-regulated phenotype of recombinant strains bearing modified sod-1 (upper panel) or rac-1 (lower panel) loci is shown on race tubes containing variable amounts of the inducer QA. (C) RT-PCR analysis of the QA-regulated expression of different loci (NCU numbers are indicated) modified by the addition of the qa-2 promoter in place of the endogenous promoter. Expression values were normalized to actin and are represented as the log₂ ratio of noninduced/WT (gray bars) and induced/WT (black bars) expression levels. QA was used at 10⁻² M. (D) Immunodetection of epitope-tagged RAC-1. Proteins were extracted from a strain containing a qa-2_p-V5-modified rac-1 locus in the absence or presence of QA (for 8 h) and from a ras-1^bd strain (indicated as WT) and blotted with anti-V5 (1:1,000). The middle panel shows the Coomassie-stained membrane (loading control), while the lower panel shows an RT-PCR for rac-1 expression, conducted for 25 cycles and with standard conditions. (E) Western blot of ras-1^bd (WT) or tagged versions of wc-2^V5H6 and frh^V5H6 decorated with the anti-V5 antibody. A Coomassie-stained blot is displayed as a loading control. (F) The V5 tag can serve as a common target for coimmunoprecipitation assays. WC-1 interacts with WC-2^V5H6 (upper panel) and FRQ interacts with FRH^V5H6 (lower panel) when the respective strains are immunoprecipitated with the anti-V5 antibody and Western blots are decorated with either polyclonal anti-WC-1 or anti-FRQ antibodies. However, no protein is detected in WT strains treated similarly. Knockout strains were included as controls for antibody specificity. The asterisks indicate nonspecific bands, while lanes labeled as T and IP correspond to totals and immunoprecipitated (IP) samples, respectively.