Department of Plant Pathology, National Chung Hsing University, Taichung, 402, Taiwan.
Transgenic Res. 2012 Apr;21(2):231-41. doi: 10.1007/s11248-011-9523-9. Epub 2011 May 20.
Tomato-infecting begomoviruses, a member of whitefly-transmitted geminivirus, cause the most devastating virus disease complex of cultivated tomato crops in the tropical and subtropical regions. Numerous strategies have been used to engineer crops for their resistance to geminiviruses. However, nearly all have concentrated on engineering the replication-associated gene (Rep), but not on a comprehensive evaluation of the entire virus genome. In this study, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting begomovirus in Taiwan, was subjected to the investigation of the viral gene fragments conferring resistance to geminiviruses in transgenic plants. Ten transgenic constructs covering the entire ToLCTWV genome were fused to a silencer DNA, the middle half of N gene of Tomato spot wilt virus (TSWV), to induce gene silencing and these constructs were transformed into Nicotiana benthamiana plants. Two constructs derived from IRC1 (intergenic region flanked with 5' end Rep) and C2 (partial C2 ORF) were able to render resistance to ToLCTWV in transgenic N. benthamiana plants. Transgenic plants transformed with two other constructs, C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3' end of the C1 ORF), significantly delayed the symptom development. Detection of siRNA confirmed that the mechanism of resistance was via gene silencing. This study demonstrated for the first time the screening of the entire genome of a monopartite begomovirus to discover viral DNA fragments that might be suitable for conferring virus resistance, and which could be potential candidates for developing transgenic plants with durable and broad-spectrum resistance to a DNA virus via a gene silencing approach.
番茄曲叶病毒，粉虱传播的双生病毒的一个成员，引起了热带和亚热带地区栽培番茄作物最严重的病毒病复合症。人们已经使用了许多策略来培育对双生病毒具有抗性的作物。然而，几乎所有这些策略都集中在工程改造 Rep 基因上，但没有对整个病毒基因组进行全面评估。在这项研究中，台湾番茄曲叶病毒（ToLCTWV），一种在台湾主要感染番茄的双生病毒，被用来研究赋予转基因植物抗双生病毒的病毒基因片段。十个包含整个 ToLCTWV 基因组的转基因构建体与沉默 DNA 融合，沉默 DNA 是番茄斑萎病毒（TSWV）N 基因的中半部分，以诱导基因沉默，并将这些构建体转化到菘蓝植物中。两个源自 IRC1（侧翼有 5'端 Rep 的基因间区）和 C2（部分 C2 ORF）的构建体能使转基因菘蓝植物对 ToLCTWV 产生抗性。转化了两个其他构建体，C2C3（C2 和 C3 ORFs 的重叠区）和 Rep2（C1 ORF 的 3'端）的转基因植物，明显延缓了症状的发展。siRNA 的检测证实了抗性机制是通过基因沉默。这项研究首次证明了筛选单分体双生病毒的整个基因组，以发现可能适合赋予病毒抗性的病毒 DNA 片段，这些片段可能是通过基因沉默方法开发具有持久和广谱抗 DNA 病毒的转基因植物的潜在候选者。
