Circular DNA plasmid in the phytopathogenic fungus Alternaria alternata: its temperature-dependent curing and...

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an approximately 5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26 degrees showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29 degrees. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23 degrees, most of which maintained the plasmid, and from mycelia grown at 29 degrees, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (approximately 1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.