RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome
RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome
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DOI：
10.1073/pnas.1212415109
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发表时间：
2012-11-20
影响因子：
11.1
通讯作者：
Lahaye, Thomas
中科院分区：
文献类型：
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作者：
Strauss, Tina;van Poecke, Remco M. P.;Lahaye, Thomas
植物病原细菌黄单胞菌属的转录激活因子样效应子（TALE）蛋白结合并转录激活宿主易感性基因，从而促进疾病。植物免疫系统已经通过进化抗性（R）基因上游的TALE结合位点来利用这种机制。例如，辣椒Bs 3和水稻Xa 27基因是由相应的TALE转录激活的过敏反应植物R基因。两种R基因都具有标志性表达模式，其中它们的转录物仅在存在而不存在相应的TALE的情况下可检测。通过使用下一代测序（RNA-seq）的转录组分析，我们测试了我们是否可以避免分离TALE诱导的R基因的费力的位置克隆。在原理验证实验中，RNA-seq用于鉴定Bs 4C的候选物，Bs 4C是来自辣椒的R基因，其介导黄单胞菌TALE蛋白AvrBs 4的识别。我们通过RNA-seq鉴定了一种主要的Bs 4C候选转录物，其仅在AvrBs 4存在下表达。互补研究证实，该候选基因对应于Bs 4C基因，并且Bs 4C启动子中的AvrBs 4结合位点指导其转录激活。Bs 4C与不能识别AvrBs 4的非功能性等位基因的比较揭示了Bs 4C启动子的TALE结合位点内的2-bp多态性。Bs 4C编码结构独特的R蛋白，并且存在于许多茄科基因组中的Bs 4C样基因似乎与辣椒Bs 4C一样受到严格调控。这些研究结果表明，TALE特异性R基因可以从大基因组作物中克隆出高效的RNA-seq方法。
Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from largegenome crops with a highly efficient RNA-seq approach.