Duplication of a Conditionally Dispensable Chromosome Carrying Pea Pathogenicity (PEP) Gene Clusters in Nectria haematococca

Abstract

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.

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

“…The nuclei of the fusion strains are clearly haploid, not diploid, the same as in the parental strains. In N. haematococca, the fused nuclei were haplodized, whereas CD chromosome accumulated more than two copies in a haploid genome (Garmaroodi and Taga 2007).…”

“…GTBM has been successfully applied in the analysis of karyotypes of several plant pathogenic fungi including A. alternata (Akamatsu et al 1999;Taga et al 1998;Taga and Murata 1994). Because of the limitation in the resolution of PFGE analysis for separating chromosomes that are close in size, combining PFGE analysis and cytological observations has been regarded as an ideal approach for karyotyping N. haematococca (Garmaroodi and Taga 2007;Taga et al 1998). Therefore, cytological visualization of mitotic chromosomes of strains from fusions between two pathotypes of A. alternata has been included in this study to evaluate the chromosomes of the hybrid strains.…”

Meiotic inheritance of a fungal supernumerary chromosome and its effect on sexual fertility in Nectria haematococca

“…Genomic DNA of fungal isolates was prepared by either PEX method described earlier (Garmaroodi & Taga 2007) or a method developed by Saitoh et al (2006).…”

“…Detection of DNA marker for each of CDCi and CDCm was carried out as described before Garmaroodi & Taga 2007). Identification of mating types was performed using degenerate primers developed by Kerenyi et al (2004).…”

“…Since accumulation in genome is a typical feature of many Bs (Camacho 2005), we examined in previous study if PDA1-CDC can be accumulated and stably maintained in genome of N. haematococca MP VI (Garmaroodi & Taga 2007). In that study, intrastrain protoplast fusion technique was used to accumulate this chromosome and was shown that two or even four copies can be accumulated and maintained in a haploid genetic background.…”