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

Abstract

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-methyldecatrienoic 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 isThe nucleotide sequence data of pcAFT-2 is available in DDBJ/ EMBL/GenBank databases under accession number AB179766 involved in the formation of the 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety of the toxin molecule.

Contribution of Peroxisomes to Secondary Metabolism and Pathogenicity in the Fungal Plant Pathogen Alternaria alternata

“…A 13 C nuclear magnetic resonance analysis of EDA purified from culture filtrates supplemented with [2-13 C]sodium acetate suggested that EDA is biosynthesized by the condensation of six molecules of acetic acid, followed by modifications, including reduction, dehydration, and decarboxylation (42). We identified AFT9, which encodes a polyketide synthase (PKS), in the AF-toxin biosynthetic gene cluster of the strawberry pathotype and found the AFT9 homologues from the Japanese pear and tangerine pathotypes by DNA gel blot analysis (51). We verified that the AFT9 ortholog of the Japanese pear pathotype, AKT9, resides in the AK-toxin biosynthetic gene cluster (S. Takaoka and T. Tsuge, unpublished data).…”

“…In addition to AFT9 and AKT9, we have identified several other genes involved in toxin production by the three pathotypes (1,15,20,34,35,36,51). They include genes common to all three pathotypes, which are probably involved in EDA biosynthesis, and genes specific to each pathotype.…”

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of Alternaria alternata

“…The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan et al 2007;Hatta et al 2002;Ito et al 2004;Johnson et al 2000;Masunaka et al 2000;Ruswandi et al 2005;Tanaka et al 1999;Tanaka and Tsuge 2000). We have shown that all strains of A. alternata pathotypes harbour small extra chromosomes that are \1.7 Mb, while nonpathogenic isolates do not have these small chromosomes (Akamatsu et al 1999).…”

“…Such a case would be advantageous for studying mechanisms underlying host-specificity of A. alternata pathotypes. The other reason for using the strawberry pathotype is that it is now the most deeply studied pathotype in A. alternata and has become a model system for the study of HST producers (Hatta et al 2002(Hatta et al , 2006Ruswandi et al 2005). The genetic constitution of the hybrid strains have also been analyzed by sequencing of marker genes derived from each parental strain.…”

“…Assays for pathogenicity and toxin production Pathogenicity and toxin production of the parental and fusion strains of A. alternata were assessed as described previously (Akamatsu et al 1997;Maekawa et al 1984;Ruswandi et al 2005;Yamagishi et al 2006). In brief, inoculated host leaves were examined for lesions 2 to 4 days after being sprayed with a spore suspension.…”