A conserved Zn2Cys6 transcription factor, identified in a spontaneous mutant from in vitro passaging, is involved in pathogenicity of the blackleg fungus Leptosphaeria maculans

Abstract Continuous passaging of microbes in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of strains, making them different from the original isolates. However, the identification of such genetic alterations is rare in studies of fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same Agrobacterium tumefaciens mediated transformation experiment, had independent T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the T-DNA insertions showed co-segregation in progeny from crosses with the reduction in pathogenicity. The genome sequences of three strains were analysed, and a point mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn2(II)Cys6 transcription factor. Homologs of this gene are found in other ascomycetes, and are also required for the pathogenicity on plants by Fusarium graminearum, Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores the mutant strains to normal pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of plant pathogenicity.