Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering-NIBGE, PO Box 577, Jhang Road, Faisalabad, Pakistan.
Virol J. 2011 Nov 2;8:499. doi: 10.1186/1743-422X-8-499.
RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.
Here a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants.
The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.
RNA 干扰（RNAi）是一种同源依赖性基因沉默机制，已被广泛用于工程植物对 RNA 病毒的抗性。然而，其在传递抗植物 DNA 病毒家族 Geminiviridae 的能力仍存在争议。尽管 RNAi 方法已在瞬时测定中显示出对单子叶植物感染的 Mastrevirus 属的 geminiviruses 有用，但尚未研究其作为传递抗双子叶植物感染的 Mastrevirus 的手段。鹰嘴豆斑驳矮化巴基斯坦病毒（CpCDPKV）是一种感染豆科植物的 Mastrevirus，会影响巴基斯坦的鹰嘴豆和其他豆科作物。
这里生产了一种发夹（hp）RNAi 构建体，该构建体包含 CpCDPKV 的复制相关蛋白基因、内含子区和部分运动蛋白基因的序列，受 Cauliflower mosaic virus 35S 启动子的控制，并稳定转化到 Nicotiana benthamiana 中。携带发夹构建体的植物受到 CpCDPKV 的挑战。所有非转基因 N. benthamiana 植物在接种后两周内都出现了 CpCDPKV 感染的症状。相比之下，接种的转基因植物均未出现感染症状，也无法通过 Southern 杂交检测到病毒 DNA。实时定量 PCR 分析鉴定出接种的转基因植物中病毒 DNA 的低水平积累。
呈现的结果表明，基于 RNAi 的抗性策略可有效保护植物免受双子叶植物感染的 Mastrevirus 的侵害。在远离接种部位的转基因植物组织中检测到的病毒水平非常低，表明病毒运动和/或病毒复制受到损害，导致植物没有明显的病毒感染迹象。
