Duplication of a conditionally dispensable chromosome carrying pea pathogenicity (PEP) gene clusters in Nectria haematococca

A supernumerary chromosome called a conditionally dispensable chromosome (CDC) is essential for pathogenicity of Nectria haematococca on pea. Among several CDCs discovered in N. haematococca, the PDA1 CDC that harbors the pisatin demethylation gene PDA1 is one of the best-studied CDCs and serves as a model for plant-pathogenic fungi. Although the presence of multiple copies is usual for supernumerary chromosomes in other eukaryotes, this possibility has not been examined well for any CDCs in N. haematococca. In this study, we produced strains with multiple copies of the PDA1 CDC by protoplast fusion and analyzed dosage effects of this chromosome. Using multiple methods, including cytological chromosome counting and fluorescence in situ hybridization, the fusion products between two transformants derived from the same strain that bears a single PDA1 CDC were shown to contain two PDA1 CDCs from both transformants and estimated to be haploid resulting from the deletion of an extra set or sets of A chromosomes in the fused nuclei. In phenotype assays, dosage effects of PDA1 CDC in the fusion products were evident as increased virulence and homoserine-utilizing ability compared with the parents. In a separate fusion experiment, PDA1 CDC accumulated up to four copies in a haploid genome.