Instituto de Biotecnología, CICVyA, CNIA, INTA, Buenos Aires, Argentina.
Virus Res. 2013 Jul;175(1):64-70. doi: 10.1016/j.virusres.2013.04.007. Epub 2013 Apr 23.
Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse genetic approach.
棉蓝病是南美洲南部棉花最重要的病毒性疾病。其病原体是棉花卷叶矮缩病毒（CLRDV），它只能通过蚜虫媒介（棉蚜）传播到宿主植物，任何试图将该病毒机械接种到宿主植物中的尝试都失败了。这一限制阻碍了对该病毒及其引起的疾病的研究。在本研究中，构建了 CLRDV 的全长 cDNA，并在花椰菜花叶病毒 35S 启动子的控制下在体内表达。开发了一种用于 CLRDV 克隆 cDNA 构建体的农杆菌介导接种系统。Northern 和免疫印迹分析表明，几个星期后，农杆菌递送的 CLRDV 复制子在棉株中引起系统性感染和典型的蓝色病症状，这与病毒 RNA 和 P3 衣壳蛋白的存在相关。我们还证明，在 agroinfected 植物中积累的病毒可以通过媒介 A. gossypii 传播。这一结果证实了具有生物活性的可传播病毒粒子的产生。此外，该克隆在侵染性的 Nicotiana benthamiana 植物中具有感染性，这些植物在接种后三周表现出叶脉间褪绿症状，并且在接种和系统叶片中均检测到 CLRDV。在拟南芥植物上进行 agroinfection 的尝试并不成功。尽管没有观察到症状，但在一些 agroinfiltrated 植物的系统叶片中，不定时检测到 P3 衣壳蛋白以及基因组和亚基因组 RNA。感染效率低表明，拟南芥是 CLRDV 的不良宿主。这是第一个关于构建具有生物活性的传染性全长棉花 RNA 病毒克隆的报告，该报告显示成功 agroinfection 了宿主和非宿主植物。本文开发的系统将有助于使用反向遗传学方法研究 CLRDV 病毒功能和植物-病毒相互作用。
