Gene inactivation in the citrus pathogenic fungus Alternaria alternata defect at the Ku70 locus associated with non-homologous end joining

Abstract

Low frequency of targeted gene disruption in filamentous fungi has been attributed to the predominance of ectopic integration that is controlled by a non-homologous end-joining (NHEJ) DNA repair mechanism. It has been shown in a number of fungi that suppression of NHEJ by inactivating the yeast Ku70 or Ku80 homolog facilitates homologous recombination, thereby enhancing the frequency of gene targeting. A Ku70 gene homolog was cloned from the necrotrophic fungus Alternaria alternata, causing citrus brown spot. The cloned gene, designated AaKu70, was independently inactivated by insertion of an acetolactate synthase gene (SUR) conferring sulfonylurea resistance or of a bacterial phosphotransferase gene (HYG) cassette conferring hygromycin resistance. The AaKu70 deficient strain reduced conidial formation and pigmentation slightly, but unchanged in toxin production and pathogenicity. Unlike many fungal species, mutation of the AaKu70 gene by inserting either SUR or HYG did not facilitate gene disruption, as tested for disruption targeting an AaAP1gene encoding a redox responsive transcription regulator or an AaHSK1 gene encoding a histidine kinase. However, a split marker approach, using truncated DNA fragments overlapping within the HYG, but not SUR, selectable marker, enhanced the frequency of AaAP1 disruption considerably in the progenitor and the ∆AaKu70 strain. Compared to the disruption targeting the AaAP1 gene, the frequency for recovery of the AaHSK1 impaired mutant was much lower, indicating an allele-dependent disruption. The present study indicates that AaKu70 has little or no effect on gene disruption and homologous integration in A. alternata.