Expression Profiles of Genes Encoded by the Supernumerary Chromosome Controlling AM-Toxin Biosynthesis and Pathogenicity in the Apple Pathotype of Alternaria alternata

Abstract

The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. Previously, we cloned two genes, AMT1 and AMT2, required for AM-toxin biosynthesis and found that these genes are encoded by small, supernumerary chromosomes of <1.8 Mb in the apple pathotype strains. Here, we performed expressed sequence tag analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO8984. A cDNA library was constructed using RNA from AM-toxin-producing cultures. A total of 40,980 clones were screened with the 1.4-Mb chromosome probe, and 196 clones encoded by the chromosome were isolated. Sequence analyses of these clones identified 80 unigenes, including AMT1 and AMT2, and revealed that the functions of 43 (54%) genes are unknown. The expression levels of the 80 genes in AM-toxin-producing and nonproducing cultures were analyzed by real-time quantitative polymerase chain reaction (PCR). Most of the genes were found to be expressed in both cultures at markedly lower levels than the translation elongation factor 1-alpha gene used as an internal control. Comparison of the expression levels of these genes between two cultures showed that 21 genes, including AMT1 and AMT2, were upregulated (>10-fold) in AM-toxin-producing cultures. Two of the upregulated genes were newly identified to be involved in AM-toxin biosynthesis by the gene disruption experiments and were named AMT3 and AMT4. Thus, the genes upregulated in AM-toxin-producing cultures contain ideal candidates for novel AM-toxin biosynthetic genes.

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

“…Host-selective toxin biosynthetic genes of A. alternata pathotypes appeared to be clustered on small chromosomes of Ͻ2.0 Mb in most of the strains tested (2,3,13,15,20,21,33,35). We demonstrated that small chromosomes that contain toxin biosynthetic genes from the strawberry, apple, and tomato pathotypes are conditionally dispensable (CD) chromosomes (2,15,21).…”

Multiple copies of AMT2 are prerequisite for the apple pathotype of Alternaria alternata to produce enough AM-toxin for expressing pathogenicity

“…We have identified four genes, AMT1, AMT2, AMT3, and AMT4, involved in AM-toxin biosynthesis (Johnson et al 2000;Ito et al 2004;Harimoto et al 2007). AMT1 encodes a 479 kDa nonribosomal peptide synthetase containing four catalytic domains responsible for the activation of each residue in AM-toxin (Fig.…”

“…1b) (Johnson et al 2000). AMT3 and AMT4 encode proteins with significant similarity to cytochrome P450 monooxygenases and thioesterase domains of nonribosomal peptide synthetase, respectively, from fungi (Harimoto et al 2007). In the apple pathotype strains, these genes were found to reside on small, conditionally dispensable (CD) chromosomes, which are not required for growth, but do confer an advantage for colonizing certain ecological niches (Covert 1998;Miao et al 1991).…”

“…5a). We previously found that IFO8984 also has three copies of AMT3 and that the twocopy mutants preserved pathogenicity (Harimoto et al 2007). Pathogenicity of the two-copy mutants of AMT2 was compared with that of the wild type or the AMT3 mutants.…”

“…We also used GD1_35s-1 and GD1_35d-1, which were the singleand two-copy mutants of AMT3, respectively, made from IFO8984 (Harimoto et al 2007). Strains were routinely maintained on potato dextrose agar (PDA, Difco, Detroit, MI, USA).…”