The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus

RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component-proteinase (HC-Pro), a viral suppressor of silencing. The studied lines carry either a tail-to-tail inverted repeat, the T4-IR transgene locus, or one of two different amplicon transgene loci encoding replication-competent viral RNA. We find that the T4-IR locus, like many sense-transgene-silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC-Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC-Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.     

RNA silencing movement in plants

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.     

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the recovery type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.     

Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing

A green fluorescent protein (GFP) transgene under the control of the 35S cauliflower mosaic virus (CaMV) promoter was introduced by Agrobacterium-mediated transformation into Nicotiana benthamiana to generate fourteen transgenic lines. Homozygous lines that contained one or two copies of the transgene showed great variation of GFP expression under ultraviolet (UV) light, which allowed classification into three types of transgenic plants. Plants from more than half of the transgenic lines underwent systemic RNA silencing and produced short interfering RNA (siRNA) as young seedlings, while plants of the remaining lines developed, in a spontaneous manner, defined GFP-silenced zones on their leaves, mostly in the form of circular spots that expanded to about 4-7 mm in size. In some of the latter lines, the GFP-silenced spots remained stable, but no systemic silencing occurred. Here we characterize this phenomenon, which we term spontaneous short-range silencing (SSRS). Biochemical analysis of silenced spot tissue did not reveal detectable levels of siRNA. However, agro-infiltration with the suppressor proteins P19 of cymbidium ring spot virus (CymRSV), HC-Pro of tobacco etch virus (TEV), and crosses to a P19 transgenic line, nevertheless suggests that low concentrations of siRNA may have a functional role in the locally silenced zone. We propose that small alterations in the steady-state concentration of siRNAs and their cognate mRNA are decisive with regard to whether silencing remains local or spreads in a systemic manner.     

RNA:诱导基因沉默

在生物体中,双链RNA(double-strand RNA,dsRNA)裂解后的小RNA可以诱导细胞质和基因组水平外源基因沉默。所谓基因沉默(gene silencing)是指生物体中特定基因由于种种原因不表达。小RNA能诱导互补信使RNA在转录后降解。RNA沉默是基因组水平的免疫现象,代表了进化过程中原始的基因组对抗外源基因序列表达的保护机制,在动植物进化中起着重要作用,RNA沉默具有抵抗病毒入侵、抑制转座子活动等作用,并调控蛋白编码基因的表达,具有十分诱人的应用前景。     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

病毒诱导的基因沉默技术用于植物色素代谢机制的研究进展

色彩是评价园艺植物观赏性状的重要指标,而植物色素是影响植物色彩表型的关键因子。植物色素及其代谢产物在植物观赏器官颜色形成、植株生长发育调节及对逆境胁迫的响应等方面发挥着重要的作用,是植物研究领域长期关注的热点问题。病毒诱导基因沉默(virus-induced gene silencing,VIGS)是利用植物同源依赖性防御机制,特异性降低宿主内源性基因表达的一种重要基因组学工具,能够通过快速诱导植物基因沉默表型的产生,表征基因的功能,为缺乏遗传转化体系的植物的基因功能鉴定提供高效可行的替代方案。本文综述了VIGS技术在植物色素的生物合成、降解和调控机制上的应用现状,并探讨了VIGS技术在探究色素调控机制上的潜力和未来前景,以期进一步完善对不同植物色素的代谢过程和调控机制的理解,为改良植物色彩性状提供参考依据。     

Dynamics of gene silencing by RNA interference

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.