A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

Abstract

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short ( approximately 250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5' and 3' flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.

Identification of autophagy as a longevity-assurance mechanism in the aging modelPodospora anserina

“…These different phenotypic characteristics were fully restored by complementation with a wild-type copy of the Paatg1 gene 34 . In our study, we constructed an independent Paatg1 deletion strain based on a protocol described in Hamann et al 12 (Fig. S2A) and observed similar phenotypic characteristics of the mutant as described earlier (Fig.…”

“…Aging of individuals can be followed macroscopically, since hyphal morphology changes during senescence of this fungus. P. anserina is accessible to experimental manipulation both by classical genetics as well as by genetic engineering 10 - 12 . The consequences of such manipulations can be analyzed by established techniques 13 .…”

Unmasking a temperature-dependent effect of theP. anserinai-AAA protease on aging and development

“…In order to investigate the role of PaIAP as a component of the mitochondrial quality control system for aging of P. anserina, we generated a strain in which its open reading frame was replaced by a phleomycin resistance cassette (ble). 35 The successful gene exchange was confirmed in phleomycin-resistant transformants by DNA gel blot analyses using PaIap-and ble-specific probes ( Fig. 2A and B).…”

Cyclophilin D links programmed cell death and organismal aging in Podospora anserina

“…Toward this goal, we set out to modulate the abundance of the protein in the mitochondrial matrix by construction of both PaCypD deletion and overexpression strains. Deletion strains were generated via replacement of PaCypD by a bifunctional selection marker gene (Hamann et al, 2005) (Fig. S2a,b).…”

“…Deletion of PaCypD in wild-type strain 's' (mating type) was performed according to a previously described method (Hamann et al, 2005). Briefly, small flanking regions of PaCypD were amplified using the 5¢-flank oligonucleotides KOCyclo1 (5¢-TTGGTACCCGTCTCAACTCTTCCTCC-3¢) and KOCyclo2 (5¢-GGAAGCTTGGTTAGTGCTCCACATGG-3¢), introducing KpnI and HindIII restriction sites and the 3¢-flank oligonucleotides KOCyclo3 (5¢-GGGGACTAGTTCGGAACGAAGGAGGGTG-3¢), and KOCyclo4 (5¢-TTGCGGCCGCCGGAAGAGCGCGACTTTG-3¢) introducing BcuI and NotI restriction sites.…”