Mutants of Nectria haematococca created by a site-directed chromosome breakage are greatly reduced in virulence toward pea

Pathogenicity of Nectria haematococca toward Pisum sativum is determined by a 1.6 million base pair (Mb) supernumerary chromosome. Repeats of the simple telomeric DNA sequence (TTAGGG) and a gene (PDA1) from the 1.6-Mb chromosome were used to direct a chromosomal break at the PDA1 locus. Mutant strains contained a novel 1.5-Mb chromosome that appeared to result from an approximately 100-kb deletion from the 1.6-Mb chromosome. These transformants (but not others that retain an apparently intact and mitotically stable 1.6- Mb chromosome) were essentially nonpathogenic on common pea, a host for the original isolate. Mutants containing the 100-kb deletion grow at a rate similar to the original strain in culture and retain the ability to colonize host tissue. Based on these results, our hypothesis is that genetic determinants of pathogenicity toward pea reside on the deleted portion of the 1.6-Mb chromosome. To our knowledge, this is the first reported use of site- directed chromosome breakage in filamentous fungi. Telomere-mediated chromosome breakage may speed genetic analysis of asexual fungi and be valuable for determining the consequences of duplicated and/or unique dispensable chromosomes in other pathogenic fungi.