Function of Genes Encoding Acyl-CoA Synthetase and Enoyl-CoA Hydratase for Host-Selective ACT-Toxin Biosynthesis in the Tangerine Pathotype of Alternaria alternata

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.