A branched pathway for transgene-induced RNA silencing in plants

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.     

A tobacco calmodulin-related protein suppresses sense transgene-induced RNA silencing but not inverted repeat-induced RNA silencing

A tobacco calmodulin-related protein, rgs-CaM, interacts with viral suppressors of RNA silencing and modulates host RNA silencing. Plants overexpressing the rgs-CaM gene were crossed with plants exhibiting sense transgene-induced RNA silencing (S-PTGS) or inverted repeat-induced RNA silencing (IR-PTGS). S44 plants harboring a sense transgene encoding a tobacco microsomal ω-3 fatty acide desaturase (NtFAD3) exhibited the S-PTGS phenotype. The frequency of the S-PTGS phenotype incidence was nearly 100 % in the hemizygous S44 plants, but was reduced to 30 % in crossbred plants with an rgs-CaM-overexpressing transgenic line. The remaining 70 % of crossbred plants successfully overexpressed the NtFAD3 transgene, and the amount of NtFAD3 small interfering RNAs (siRNAs) was largely decreased. In contrast, overexpression of rgs-CaM did not suppress siRNA production in the IR-PTGS that targeted the NtFAD3 gene. These results indicated that rgs-CaM suppresses RNA silencing at a step upstream of siRNA production and does not interfere with the later steps of RNA silencing, including siRNA-mediated RNA degradation.     

RNA沉默与植物病毒

植物中RNA沉默（RNAsilencing)亦称为转录后基因沉默（PTGS）或共抑制,是植物抵抗外来核酸（转座子、转基因或病毒）入侵,并保护自身基因组完整性的一种防御机制。RNA沉默是近十年来发现的植物界中普遍存在的现象,已成为植物分子生物学领域的一个新的研究方向。对RNA沉默特点和机制的研究表明,植物病毒与（转基因）植物内发生的RNA沉默有着密切的联系,作者从病毒对RNA沉默的诱导、抑制、防御等方面,简述了RNA沉默与病毒的关系。并对病毒载体所诱导的RNA沉默在植物发育和基因组功能分析等方面的应用价值进行了讨论。     

Small RNAs from cereal powdery mildew pathogens may target host plant genes

Small RNAs (sRNAs) play a key role in eukaryotic gene regulation, for example by gene silencing via RNA interference (RNAi). The biogenesis of sRNAs depends on proteins that are generally conserved in all eukaryotic lineages, yet some species that lack part or all the components of the mechanism exist. Here we explored the presence of the RNAi machinery and its expression as well as the occurrence of sRNA candidates and their putative endogenous as well as host targets in phytopathogenic powdery mildew fungi. We focused on the species Blumeria graminis, which occurs in various specialized forms (formae speciales) that each have a strictly limited host range. B. graminis f. sp. hordei and B. graminis f. sp. tritici, colonizing barley and wheat, respectively, have genomes that are characterized by extensive gene loss. Nonetheless, we find that the RNAi machinery appears to be largely complete and expressed during infection. sRNA sequencing data enabled the identification of putative sRNAs in both pathogens. While a considerable part of the sRNA candidates have predicted target sites in endogenous genes and transposable elements, a small proportion appears to have targets in planta, suggesting potential cross-kingdom RNA transfer between powdery mildew fungi and their respective plant hosts.     

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-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants

In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.