Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata

Aiko Tanaka, Hiroshi Shiotani, Mikihiro Yamamoto, Takashi Tsuge

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone of the wild-type strain was isolated. Structural and functional analyses of an 8.0-kb region corresponding to the tagged site indicated the presence of two genes. One, designated AKT1, encodes a member of the class of carboxylactivating enzymes. The other, AKT2, encodes a protein of unknown function. The essential roles of these two genes in both AK-toxin production and pathogenicity were confirmed by transformation-mediated gene disruption experiments. DNA gel blot analysis detected AKT1 and AKT2 homologues not only in the Japanese pear pathotype strains but also in strains from the tangerine and strawberry pathotypes. The host-specific toxins of these two pathotypes are similar in structure to AK-toxin. Homologues were not detected in other pathotypes or in nonpathogenic strains of A. alternata, suggesting acquisition of AKT1 and AKT2 by horizontal transfer.

Original languageEnglish
Pages (from-to)691-702
Number of pages12
JournalMolecular Plant-Microbe Interactions
Volume12
Issue number8
Publication statusPublished - Aug 1999

Fingerprint

insertional mutagenesis
Pyrus
Alternaria
Mutagenesis
Insertional Mutagenesis
Cloning
Alternaria alternata
Biosynthesis
pathotypes
pears
Organism Cloning
molecular cloning
toxins
Genes
biosynthesis
Plasmids
Enzymes
Virulence
DNA
plasmids

ASJC Scopus subject areas

  • Microbiology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology

Cite this

Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata. / Tanaka, Aiko; Shiotani, Hiroshi; Yamamoto, Mikihiro; Tsuge, Takashi.

In: Molecular Plant-Microbe Interactions, Vol. 12, No. 8, 08.1999, p. 691-702.

Research output: Contribution to journalArticle

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