DNA transposon fossils present on the conditionally dispensable chromosome controlling AF-toxin biosynthesis and pathogenicity of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces host-specific AF-toxin and causes Alternaria black spot of strawberry. Previously, we isolated cosmid clones pcAFT-1 and pcAFT-2 from strain NAF8 of the strawberry pathotype that contain AF-toxin biosynthetic genes, named AFT genes. In a molecular characterization here of pcAFT-1 and pcAFT-2, 11 AFT genes and five transposon-like sequences, named TLS-S1 to TLS-S5, were detected. The nucleotide sequences of TLS-S1 and TLS-S4 share high homology, and their putative products have similarity to transposases of the hAT family transposons. Thus, TLS-S1 and TLS-S4 were renamed TLS-S1-1 and TLS-S1-2, respectively. Amino acid sequences deduced from TLS-S2, TLS-S3, and TLS-S5 have similarity to transposases of the Fot1/Pogo family transposons, but they are significantly different. All five sequences have incomplete open reading frames (ORFs) for transposases owing to deletions, termination codons, and/or frameshifts, indicating that they are inactivated elements. Analysis of genomic distribution of these sequences revealed that they are specifically distributed on a 1.05-Mb chromosome of NAF8, which has been identified as a conditionally dispensable (CD) chromosome encoding AFT genes. The presence of three, four, and three copies of TLS-S1, TLS-S2, and TLS-S3, respectively, and a single copy of TLS-S5 on the CD chromosome were estimated by DNA gel blot analysis. The remaining copy of TLS-S1 and the three copies of TLS-S2 were isolated and identified to also encode incomplete ORFs. Thus, it appears that all copies of the transposon-like sequences identified are inactivated elements (fossils) unique to the CD chromosome in the genome of the strawberry pathotype. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB236733 (TLS-S1-1), AB236734 (TLS-S1-2), AB236735 (TLS-S1-3), AB236736 (TLS-S2-1), AB236737 (TLS-S2-2), AB236738 (TLS-S3), and AB236739 (TLS-S5)     

Detoxification of α-tomatine by tomato pathogens Alternaria alternata tomato pathotype and Corynespora cassiicola and its role in infection

In tomato plants, α-tomatine, a steroidal glycoalkaloid saponin, inhibits fungal growth. Tomato pathogens that produce host-specific toxins, Alternaria alternata tomato pathotype causing Alternaria stem canker and Corynespora cassiicola causing Corynespora target spot, were investigated for sensitivity to α-tomatine. Although spore germination of A. alternata pathogenic and nonpathogenic to tomato and of C. cassiicola pathogenic to tomato was not affected by 0.1 mM α-tomatine, spore germination of C. cassiicola nonpathogenic to tomato was significantly inhibited. This result showed that A. alternata, regardless of its pathogenicity, and only the C. cassiicola pathogenic to tomato are resistant to α-tomatine. Germinating spores of A. alternata and C. cassiicola resistant to α-tomatine detoxified α-tomatine by degrading it to a less polar product. After inoculation of tomato leaves, spores of A. alternata and C. cassiicola nonpathogenic to tomato germinated and formed appressoria, but did not form infection hyphae in host tissues. When a host-specific toxin (CCT-toxin) produced by C. cassiicola pathogenic to tomato was added to nonpathogenic spores, colonization within leaves was observed in A. alternata, but not in C. cassiicola. On the other hand, when spores of C. cassiicola nonpathogenic to tomato were suspended in spore germination fluid of nonpathogenic A. alternata with α-tomatine detoxification activity, the fungus could be induced to colonize leaves in the presence of CCT-toxin. These results indicate that A. alternata tomato pathotype and C. cassiicola pathogenic to tomato detoxify α-tomatine during infection and that this detoxification is essential for host colonization by pathogens that produce host-specific toxins.     

Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.     

Identification of putative defense-related genes in Japanese pear against <Emphasis Type="Italic">Alternaria alternata</Emphasis> infection using suppression subtractive hybridization and expression analysis

The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.