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
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
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DOI：
10.1007/s10327-004-0170-3
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发表时间：
2005-04-05
影响因子：
1.2
通讯作者：
Yamamoto, Mikihrio
中科院分区：
文献类型：
--
作者：
Ruswandi, Sheila;Kitani, Keiji;Yamamoto, Mikihrio
互隔交链孢菌（Alternaria alternata）的草莓致病型产生寄主特异性的AF毒素，引起草莓黑斑病。毒素基因簇中的基因目前正在鉴定和表征。在这项研究中，基因组粘粒克隆，pcAFT-2，结构特征。该粘粒含有AFT同系物，发现其参与AF毒素的生物合成。这些同源物被命名为AFTR-2和AFT 3 -2。另外还鉴定了4个新的开放阅读框（AFT 9 -1，AFT 10 -1，AFT 11 -1，AFT 12 -1）和2个转座子样序列（TLS-S4，TLS-S5）。这些ORF分别编码聚酮合酶、酰基辅酶A脱氢酶、P450单加氧酶和氧化还原酶。所有ORF的转录本均被检测到。DNA凝胶印迹分析检测到这四个ORF的同源物仅在草莓，草莓，日本梨致病型，共享一个共同的9，10-环氧-8-羟基-9-甲基癸三烯酸部分，在其毒素结构。靶向AFT 10 -1，其编码酰基辅酶A脱氢酶，产生单拷贝和双拷贝突变体，其在宿主叶片上的病变数量大大减少，同时毒素产生减少，证实了其在致病性中的作用。因此，AFT 10 -1存在于链格孢菌基因组中的多个拷贝中;并且基于梨和日本梨致病型中同源物的存在，它参与毒素分子的9，10-环氧-8-羟基-9-甲基-癸三烯酸部分的形成。
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 is involved in the formation of the 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety of the toxin molecule.