Laboratório de Biologia Celular e Molecular, CENA/USP, Avenida Centenário, 303 CP 96 Piracicaba-SP, 13400-970, Brazil.
Mol Plant Pathol. 2003 Sep 1;4(5):327-35. doi: 10.1046/j.1364-3703.2003.00181.x.
SUMMARY This is the first report of a genotypic analysis of the phytopathogenic bacteria Xylella fastidiosa (Xf) using differences within intra- and intergenic regions of pathogenic genes. Orthologous sequences from the genome of Xf were identified for genes involved in the regulation of pathogenicity factors (rpf) from Xanthomonas campestris pv. campestris (Xcc). While the rpf genes were conserved, the chromosomal region revealed differences in gene sizes and intergenic spacings and a major translocational event when compared to Xcc. Primers were designed to amplify three regions: the intragenic region of rpfA (2354 bp), the intergenic region between rpfA and rpfB (5772 bp), and the intergenic region between rpfC and rpfF (2314 bp). Amplicons were obtained for all three regions from 32 of the 33 Xf isolates tested from citrus, grape, coffee, plum, hibiscus and periwinkle. Three Xcc isolates from cruciferous plants only generated PCR products for the rpfC-F region. Cleaved amplified polymorphic sequences (CAPS) (Taq(alpha)I) revealed differential banding profiles for the rpfA-B and rpfC-F regions. Xylella isolates were separated into seven groups via rpfA-B, of which five contained only citrus, while the other two had citrus, grape and coffee, and citrus, coffee, plum and hibiscus isolates. rpfC-F separated the isolates into three host-related groups. Citrus, coffee and hibiscus isolates formed one group, while the other two groups were comprised solely of grape and plum isolates. Xcc isolates formed an out-group. In silico analysis supports these results, which reveal the potential of the rpf genes for genotypic analysis of Xylella fastidiosa.
摘要 这是首次利用致病性基因的种内和种间区域差异对黄单胞菌属植物病原菌 Xylella fastidiosa（Xf）进行基因分析。本研究鉴定了 Xf 基因组中与黄单胞菌野油菜致病变种（Xcc）致病因子调节基因（rpf）同源的序列。虽然 rpf 基因具有保守性，但与 Xcc 相比，该基因的染色体区域在基因大小和基因间隔方面存在差异，并且存在一个主要的转位事件。设计了用于扩增三个区域的引物：rpfA 的内含子区（2354bp）、rpfA 和 rpfB 之间的基因间隔区（5772bp）以及 rpfC 和 rpfF 之间的基因间隔区（2314bp）。从柑橘、葡萄、咖啡、李树、芙蓉和长春花中测试的 33 个 Xf 分离株中，所有三个区域均获得了扩增子。来自十字花科植物的三个 Xcc 分离株仅生成 rpfC-F 区域的 PCR 产物。切割扩增多态性序列（CAPS）（Taq（alpha）I）显示 rpfA-B 和 rpfC-F 区域的差异带型谱。rpfA-B 将 Xylella 分离株分为七个组，其中五个仅包含柑橘，而另外两个则包含柑橘、葡萄和咖啡，以及柑橘、咖啡、李树和芙蓉。rpfC-F 将分离株分为三个与宿主相关的组。柑橘、咖啡和芙蓉分离株形成一组，而另外两组仅由葡萄和李树分离株组成。Xcc 分离株形成一个外群。计算机分析支持这些结果，这些结果表明 rpf 基因具有分析 Xylella fastidiosa 基因型的潜力。
