Structural analysis of cosmid clone pcAFT-2 carrying AFT10-1 encoding an acyl-CoA dehydrogenase involved in AF-toxin production in the strawberry pathotype of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces the host-specific AF-toxin and causes black spot of strawberry. The genes in the toxin gene cluster are currently being identified and characterized. In this study, a genomic cosmid clone, pcAFT-2, was structurally characterized. This cosmid contains AFT homologs, which were found to be involved in AF-toxin biosynthesis. These homologs were designated AFTR-2 and AFT3-2. Four new open reading frames (ORFs) (AFT9-1, AFT10-1, AFT11-1, AFT12-1) and two transposon-like sequences (TLS-S4, TLS-S5) were also identified. These ORFs were shown to encode for polyketide synthase, acyl-CoA dehydrogenase, P450 monooxygenase, and an oxidoreductase, respectively. Transcripts of all the ORFs were detected. DNA gel blot analysis detected homologs of these four ORFs only in the tangerine, strawberry, and Japanese pear pathotypes, which share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety in their toxin structure. Targeting of AFT10-1, which encodes an acyl-CoA dehydrogenase, produced single- and double-copy mutants with highly reduced numbers of lesions on host leaves concomitant with reduced toxin production, confirming its role in pathogenicity. Thus, AFT10-1 exists in multiple copies in the genome of Alternaria alternata; and based on the presence of homologs in the tangerine and Japanese pear pathotypes, it is involved in the formation of the 9,10-epoxy-8-hydroxy-9- methyl-decatrienoic acid moiety of the toxin molecule.