A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.
A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.
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DOI：
10.1371/journal.ppat.1002021
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发表时间：
2011-05
期刊：
影响因子：
6.7
通讯作者：
Masuta C
中科院分区：
文献类型：
--
作者：
Shimura H;Pantaleo V;Ishihara T;Myojo N;Inaba J;Sueda K;Burgyán J;Masuta C
受病毒感染的植物上的症状通常对给定的病毒非常特异。参与病毒症状诱导的分子机制已被广泛研究，但仍然知之甚少。黄瓜花叶病毒（Cucumber mosaic virus，CMV）Y卫星RNA（Y-sat）是一种非编码的亚病毒RNA，能改变CMV在特定寄主上引起的典型症状，在其天然寄主烟草上引起亮黄色的花叶。Y-sat诱导的黄色花叶未能在感染的拟南芥和番茄植株中发展，这表明Y-sat与其宿主之间存在非常特异的相互作用。在这项研究中，我们发现Y-sat产生特异性短干扰RNA（siRNA），干扰宿主基因，从而诱导特定症状。我们发现烟草镁原卟啉螯合酶亚基I（ChlI，参与叶绿素合成的关键基因）的mRNA具有与Y-sat序列互补的22-nt序列，包括4个G-U对，并且病毒感染植物中的Y-sat衍生的siRNA通过靶向互补序列下调ChlI的mRNA。ChlI mRNA在表达Y-sat反向重复序列的转基因株系中也下调。引人注目的是，修改Y-sat序列，以恢复22-nt的互补性，拟南芥和番茄ChlI mRNA导致黄化症状，在Y-sat感染的拟南芥和番茄，分别。在5′-RACE实验中，ChlI转录本在22-nt互补序列的预期中间位置被切割。在使用农杆菌渗透的GFP传感器实验中，我们进一步证明了Y-sat特异性靶向含有ChlI的22-nt互补序列的传感器mRNA。我们的研究结果提供了直接的证据，即所鉴定的来自病毒卫星RNA的siRNA通过基于RNA沉默的宿主基因调控直接调节病毒疾病症状。 黄瓜花叶病毒（Cucumber mosaic virus，CMV）Y卫星RNA（Y-satellite RNA，Y-sat）是一种有趣的亚病毒RNA，它能将CMV诱导的烟草绿色花叶转变为亮黄色花叶。病毒诱导症状的分子基础还不清楚，这种Y-sat介导的症状修饰一直是一个长期存在的谜团。在本研究中，我们发现了参与Y-sat诱导的泛黄的分子机制。首先，我们证明了转基因N.表达Y-sat反向重复序列的本塞姆氏植物也产生了黄色表型，与Y-sat感染的植物相似。结果表明，烟草镁原卟啉螯合酶亚基I基因（ChlI，叶绿素合成的关键基因）在转基因植株和感染Y-sat的植株中表达下调。然后，我们确定了一个22个核苷酸长的序列，是互补的Y-sat包括四个G-U对在ChlI mRNA。最后，我们证明了来自Y-sat的短干扰RNA（siRNA）特异性靶向并下调ChlI mRNA，从而损害叶绿素生物合成途径。Y-sat诱导的症状修饰的分子基础的这一发现首次证明了亚病毒RNA可以通过RNA沉默机制调节宿主基因表达来诱导疾病症状。
Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5′-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is an interesting subviral RNA because it changes the green mosaic induced by CMV into a bright yellow mosaic in Nicotiana tabacum. The molecular basis underlying the induction of symptoms by viruses is not well understood, and this Y-sat-mediated modification of symptoms has been a long-standing mystery. In this study, we discovered the molecular mechanism involved in the Y-sat-induced yellowing. First, we showed that transgenic N. benthamiana plants that expressed the inverted-repeat sequence of Y-sat also developed a yellow phenotype, similar to the Y-sat-infected plants. Then, we found that tobacco magnesium protoporphyrin chelatase subunit I gene (ChlI, the key gene involved in chlorophyll synthesis) was downregulated in the transgenic plants and in the Y-sat-infected plants. We then identified a 22-nt long sequence that is complementary to the Y-sat including four G-U pairs in the ChlI mRNA. Finally, we demonstrated that a short interfering RNA (siRNA) derived from Y-sat specifically targeted and downregulated the ChlI mRNA, thus impairing the chlorophyll biosynthesis pathway. This discovery of the molecular basis of the symptom modification induced by Y-sat is the first demonstration that a subviral RNA can induce disease symptoms by regulating host gene expression through the RNA silencing machinery.
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