Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end- joining-deficient genetic background

Abstract

The akuA gene encoding the Ku70 component of the nonhomologous end-joining machinery was deleted in the opportunistic pathogen Aspergillus fumigatus. No obvious phenotype could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were increased as deduced from targeting the laccase-encoding abr2 gene.

FIG. 1.

Aspergillus fumigatus genome includes a ku70 orthologue. (A) Structure of the genomic A. fumigatus akuA gene and the deleted akuA::ptrA locus of strain AfS28. Recognition sites for restriction enzymes as employed in Southern analyses are indicated; the positions of 5′- and 3′-specific probes are given as black bars. (B) Autoradiographs from Southern membranes. Genomic DNA from strains D141 (wild type) and AfS28 (akuAΔ) was subjected to digestion with enzymes BamHI (Ba), BclI (Bc), SphI (Sp), NcoI (Ni), and NsiI (Ns), and separated by agarose gel electrophoresis. After blotting and immobilization, membranes were hybridized with a radioactively labeled 5′-specific (left panel) or 3′-specific (right panel) probe as specified in panel A; M indicates the DNA size standard with fragments' lengths given on either edge. (C) Assessment of strain AfS28 in an insect virulence model. Groups of 15 larvae of the greater wax moth Galleria mellonella were infected with 8 × 10⁶ conidia from D141 and AfS28, respectively, by injection of a 20-μl conidial suspension in saline supplemented with 10 μg/ml rifampin in the left last proleg. Larvae were kept in the dark at 30°C and inspected on a daily basis for motility and melanization.

FIG. 2.

Targeting the A. fumigatus abr2 locus to assess frequencies of homologous recombination in a nonhomologous end-joining-deficient background. (A) Genomic architecture of the A. fumigatus abr2 locus before and after replacement with the marker cassette of pME3003. The black bar gives the position of the hybridization probe in Southern experiments with restriction sites used as indicated. (B) Autoradiograph of Southern hybridization experiment with transformants derived from D141 and AfS28, respectively. Genomic DNA prepared from the specified strains was digested with restriction enzymes EcoRV (EV) or PstI (PI); M stands for the DNA size standard with fragments' sizes indicated. (C) Appearance of an A. fumigatus abr2Δ strain (right) with its brownish conidia in comparison to the wild-type progenitor (left) displaying the typical gray-green spore pigmentation.