Pang S Z, Jan F J, Gonsalves D
Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8261-6. doi: 10.1073/pnas.94.15.8261.
RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic Nicotiana benthamiana plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387-453 bp) of the N gene displayed resistance through post-transcriptional gene silencing. Although smaller N gene segments (92-235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (i) a critical length of N transgene (236-387 bp) is required for a high level of transgene expression and consequent gene silencing, and (ii) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.
RNA介导的病毒抗性最近被证明是转基因植物中转录后转基因沉默的结果。本研究旨在表征转基因长度和非靶标DNA序列对转基因植物中RNA介导的番茄斑萎病毒抗性的影响。构建了转基因本氏烟草植株，以表达番茄斑萎病毒（TSWV）核衣壳（N）蛋白的不同区域。表达N基因半基因片段（387 - 453 bp）的转基因植物通过转录后基因沉默表现出抗性。虽然较小的N基因片段（92 - 235 bp）单独在转基因植物中表达时不能赋予抗性，但这些片段与非靶标绿色荧光蛋白基因DNA融合时可赋予抗性。这些结果表明：（i）高水平的转基因表达及随后的基因沉默需要N转基因的临界长度（236 - 387 bp）；（ii）转录后基因沉默机制可以反式失活与短至110 bp的同源转基因片段的入侵番茄斑萎病毒基因组。因此，转录后转基因沉默的激活需要比入侵病毒基因组反式失活所需的转基因大得多。这些结果增加了开发一种在转基因植物中设计多重病毒抗性的简单新策略的可能性。
