Geminivirus-mediated gene silencing from Cotton leaf crumple virus is enhanced by low temperature in cotton

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.     

Plant Mobile Small RNAs

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. One of the most fascinating aspects of RNA silencing found in plants and invertebrates is its mobile nature—in other words, its ability to spread from the cell where it has been initiated to neighboring cells. This phenomenon relies on the movement of small noncoding RNA molecules (sRNA, 21–24 nucleotides [nt] in length) that provide the sequence specificity of the silencing effects. In plants, there are two major classes of sRNAs: short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These sRNAs are generated by diverse and sometimes interacting biochemical pathways, which may influence their mobility. Movement of plant sRNAs falls into two main categories: cell-to-cell (short-range) and systemic (long-range) movement (Melnyk et al. 2011).     

RNA沉默信号分子研究进展

RNA沉默是一种由21．26nt RNA分子介导的真核生物体内普遍存在的以序列特定性行为抑制基因表达的保守途径。RNA沉默最显著的特征在于它是一种非细胞自发的过程,即在植物和小杆线虫（Caenorhabditis elegans）中RNA沉默均能被局部诱导,随后将沉默状态传导至整个生物体。RNA沉默的系统传导特性表明,RNA沉默途径中存在着可移动的信号分子。就RNA沉默信号传导的特征和可能的信号分子等做一综述。     

Intercellular and systemic movement of RNA silencing signals

In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.     

Viral induction and suppression of RNA silencing in plants

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

Silencing of a meristematic gene using geminivirus-derived vectors

Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome-mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down-regulated throughout meristematic tissues. Our results demonstrate that geminivirus-derived vectors can be used to study genes involved in meristem function in intact plants.     

RNA silencing of single and multiple members in a gene family of rice

RNA silencing with inverted repeat (IR) constructs has been used to suppress gene expression in various organisms. However, the transitive RNA-silencing effect described in plants may preclude the use of RNA silencing for a gene family. Here, we show that, in rice (Oryza sativa), transitive RNA silencing (spreading of double-stranded RNA along the target mRNA) occurred with the green fluorescent protein transgene but not with the endogenous phytoene desaturase gene. We fused IR copies of unique 3' untranslated regions derived from the rice OsRac gene family to a strong promoter and stably introduced them into rice. Each of the seven members of the OsRac gene family was specifically suppressed by its respective IR construct. We also examined IR constructs in which multiple 3' untranslated regions were fused and showed that three members of the OsRac gene family were effectively suppressed by a single construct. Using highly conserved regions of the two members of the OsRac gene family, we also suppressed the expression of all members of the gene family with variable efficiencies. These results suggest that RNA silencing is a useful method for the functional analysis of gene families in rice and other plants.     

植物中 RNA 分子系统运输的研究进展

RNA 分子主要以单链的形式存在于生物体内,既担负着贮存及转移遗传信息的作用,又能作为核酶直接在细胞内发挥代谢功能 . 在植物中 RNA 也可作为活跃的信号分子调控基因表达和发育 . 介绍了包括病毒 RNA 、 RNA 沉默信号、特异内源 RNA 等 RNA 分子,在植物体内的系统运输及其在植物基因表达调控中所起作用的研究进展 .