State Key Laboratory for Agrobiotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, China.
Virus Res. 2015 Feb 2;197:13-6. doi: 10.1016/j.virusres.2014.12.005. Epub 2014 Dec 10.
Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes.
黄花叶病毒是布罗克病毒科黄花叶病毒属的一个新种，广泛分布于中国大陆和韩国，是一种重要的十字花科作物病毒。基于油菜、芜菁、萝卜和白菜上分离物的 6 个黄花叶病毒基因组序列，存在 3 种基因型，即 BrYV-A、BrYV-B 和 BrYV-C，它们主要在基因组的 5'端的一半不同。黄花叶病毒是一种蚜虫传播、韧皮部局限的病毒。使用传染性 cDNA 克隆是感染植物的另一种方法，允许进行反向遗传学研究。在本研究中，构建了 BrYV-A、重组 BrYV5B3A 和 BrYV-C 的全长 cDNA 克隆，这些克隆在花椰菜花叶病毒 35S 启动子的控制下。利用这些 cDNA 克隆，建立了农杆菌介导的黄花叶病毒侵染 Nicotiana benthamiana 的系统。在侵染全长 BrYV cDNA 克隆 3 天后，侵染叶片出现坏死症状，而上部叶片无明显症状。对来自上部叶片的样品进行反转录-PCR（RT-PCR）和 Western blot 检测，结果表明 BrYVs 的最大感染效率可达 100%。通过 northern 杂交进一步证实了 BrYV-A、BrYV-5B3A 和 BrYV-C cDNA 克隆的感染力。该系统的建立将有助于进一步研究黄花叶病毒，如宿主范围、致病性、病毒基因功能和植物-病毒-载体相互作用，特别是用于区分 3 种基因型的差异。
