High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora

Abstract

Gene disruption in Pestalotiopsis microspora is blocked by the formation of extrachromosomal DNAs with telomeric repeats in transformants generated using the protoplast-PEG approach. In order to avoid this condition and to provide molecular tools, a highly efficient gene targeting method, mediated by single-stranded DNA homologous disruption of Agrobacterium tumefaciens-mediated transformation (ATMT) with one-step construction of Agrobacterium-recombination-ready-plasmids (OSCAR) was established for the first time in this fungus. Conditions affecting recombination for OSCAR were optimized. The polyketide synthase gene pks1—an essential gene for melanin biosynthesis—was selected as a target gene because of the clear phenotypic difference between the pks1 mutant and the wild-type strain. Disruption of pks1 by this method resulted in 80 % albino transformants compared with black colonies of the wild-type strain. Southern analysis and PCR amplification verified that a single-copy homologous recombination event had occurred at the pks1 locus. The pattern of secondary metabolites and the production of conidia were affected by the loss of pks1. This study demonstrated that ATMT with single-stranded DNA is an effective method for gene targeting in Pestalotiopsis microspora, which should facilitate genetic modification in this species. This study also indicates that crosstalk exists between the biosynthesis of melanin and other secondary metabolites in this fungus.