Efficient tools to target DNA to Podospora anserina

Abstract

Because, in filamentous fungi, integration of transgenes proceeds mainly through nonhomologous recombination, transformation results in a random distribution of these DNA sequences. Moreover, they are often found as multiple copies clustered at a single locus. Therefore, for a given transgene, expression may be highly variable among independent transformants. To get rid of both multiple copies and position effects, due to the impact of the chromatin structure upon transgene expression, we constructed tools to specifically target single copies at two well-defined loci, Pa_2_3690 and Pa_4_5450 (Espagne et al., 2008). These genes have been chosen for the following reasons: (1) they have a relatively stable expression during the entire Podospora’s life cycle, (2) complete deletion of their ORF does not lead to an abnormal phenotype (Bidard et al., 2011), (3) they are located on two different chromosomes and (4) their second division segregation (SDS) frequency is 80% and 90%, respectively. Besides, a highly efficient homologous recombination ΔPaKu70 strain is available (El-Khoury et al., 2008), which makes gene replacement fairly easy. Taking advantage of this strain, we designed two plasmid tools to target genomic integration of any DNA fragment.