Lim P O, Ryu J S, Lee H J, Lee U, Park Y S, Kwak J M, Choi J K, Nam H G
Department of Life Science, Pohang University of Science and Technology, Korea.
Mol Cells. 1997 Jun 30;7(3):313-9.
Red pepper, one of the most important vegetable crops in Korea, is severely affected by viral diseases causing 20-50% reduction in product yield. A pepper strain of tobacco mosaic virus (TMV-p) is the most common virus in red pepper. To study the molecular structure of the TMV-p virus, we generated cDNA clones of the viral genome. Partial sequencing of a few cDNA clones revealed that TMV-p shares a 98% identity at the nucleotide level with the Spanish isolate of pepper mild mottle virus (PMMV-s). This suggests that TMV-p should be reclassified as the Korean isolate of PMMV (PMMV-k). The coat protein (CP) gene together with the 3' untranslated region of the PMMV-k virus was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligomers deduced from the sequence of PMMV-s. The sequence analysis of the CP gene and the 3' untranslated region further confirmed that PMMV-k is highly related to PMMV-s. The CP gene and the 3' untranslated region of PMMV-k were cloned into a plant expression vector and the construct was introduced into tobacco plants. The transgenic plants expressing the PMMV-k CP gene were delayed in developing systemic disease or failed to develop symptoms at all after inoculation with PMMV-k. Delay of symptoms was also observed when the plants were inoculated with TMV-OM which shares a 74% homology with PMMV-k in the amino acid sequence of the CP region. In a local lesion host, the CP expressing plants exhibited a greatly reduced number of necrotic lesions as compared to control plants after inoculation with TMV-OM. Our results show that CP-mediated viral resistance is readily applicable in the case of PMMV-k and can provide resistance to other viruses in the tobamovirus group.
辣椒是韩国最重要的蔬菜作物之一，受病毒病影响严重，导致产品产量降低20%-50%。烟草花叶病毒的一个辣椒株系（TMV-p）是红辣椒中最常见的病毒。为了研究TMV-p病毒的分子结构，我们构建了该病毒基因组的cDNA克隆。对一些cDNA克隆进行部分测序后发现，TMV-p在核苷酸水平上与辣椒轻斑驳病毒的西班牙分离株（PMMV-s）有98%的同一性。这表明TMV-p应重新分类为PMMV的韩国分离株（PMMV-k）。通过逆转录聚合酶链反应（RT-PCR），使用从PMMV-s序列推导的寡核苷酸，获得了PMMV-k病毒的外壳蛋白（CP）基因以及3'非翻译区。CP基因和3'非翻译区的序列分析进一步证实PMMV-k与PMMV-s高度相关。将PMMV-k的CP基因和3'非翻译区克隆到植物表达载体中，并将构建体导入烟草植株。表达PMMV-k CP基因的转基因植株在接种PMMV-k后，全身发病延迟或根本不出现症状。当这些植株接种与PMMV-k在CP区域氨基酸序列上有74%同源性的TMV-OM时，也观察到了症状延迟。在局部病斑宿主中，接种TMV-OM后，表达CP的植株与对照植株相比，坏死病斑数量大大减少。我们的结果表明，CP介导的病毒抗性在PMMV-k的情况下很容易应用，并且可以为烟草花叶病毒组中的其他病毒提供抗性。
