Bayer Crop Science, Woodland, California, United States of America.
Bayer Crop Science, Chesterfield Parkway, St. Louis, Missouri, United States of America.
PLoS One. 2021 Mar 15;16(3):e0245422. doi: 10.1371/journal.pone.0245422. eCollection 2021.
Gene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand topically-induced, systemic transgene silencing in Nicotiana benthamiana. A previous report details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event. This investigation revealed an inadvertent co-integration of part of a bacterial transposase in this line. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous GFP16C lines produced for this study ranged from 50-72% of the homozygous GFP16C line. GFP expression was equivalent to GFP16C in a two-copy event. Local GFP silencing was observed in all transgenic and GFP16C hemizygous lines after topical application of carbon dot-based formulations containing a GFP targeting dsRNA. The GFP16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of topically-induced, systemic transgene silencing in N. benthamiana.
利用局部 dsRNA 对植物进行基因沉默是一种新方法，具有成为未来农业生产系统可持续组成部分的潜力。然而，需要更多的研究才能使这项技术成为当前作物保护实践的经济有效补充。系统性基因沉默是一个关键的实现方面。本研究的目的是更好地理解在烟草原生质体中局部诱导的、系统性转基因沉默。以前的一份报告详细介绍了绿色荧光蛋白 (GFP) 转基因在著名的烟草原生质体 GFP16C 事件中的整合位点测序。这项研究揭示了该系中部分细菌转座酶的意外共整合。为了确定这种转基因构型对系统性沉默的影响，生产了具有或不具有转座酶序列的新 GFP 转基因系。本研究产生的 19 个单拷贝事件和 3 个半合子 GFP16C 系中的 GFP 表达水平在 50-72%之间，与纯合子 GFP16C 系相当。在含有 GFP 靶向 dsRNA 的基于碳点的制剂局部处理后，所有转基因和 GFP16C 半合子系均观察到 GFP 表达的局部沉默。只有在双拷贝系中观察到 GFP16C 样系统性沉默表型。部分转座酶对转基因表达水平、局部 GFP 沉默、小 RNA 丰度和分布或转基因系中的系统性 GFP 沉默没有影响。我们得出结论，高转基因表达水平是烟草原生质体中局部诱导、系统性转基因沉默的关键促成因素。
