Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

Abstract

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others.