Enhanced exogenous RNAi by loop-ended double-stranded RNA in plants.

Exogenous RNA interference (exoRNAi) has emerged as a promising tool for gene silencing in plants, yet a robust and efficient exoRNAi technology remains elusive. Here we evaluated a Loop-ended dsRNA (ledRNA) design for enhancing exoRNAi efficacy. ledRNA has a dumbbell-like structure, consisting of a long dsRNA stem flanked by single-stranded loops, with a nick site at the dsRNA stem.

G-U base-paired hpRNA confers potent inhibition of small RNA function in plants.

MicroRNA (miRNA) target mimicry technologies, utilizing naturally occurring miRNA decoy molecules, represent a potent tool for analyzing miRNA function. In this study, we present a highly efficient small RNA (sRNA) target mimicry design based on G-U base-paired hairpin RNA (hpG:U), which allows for the simultaneous targeting of multiple sRNAs. The hpG:U constructs consistently generate high amounts of intact, polyadenylated stem-loop (SL) RNA outside the nuclei, in contrast to traditional hairpin RNA designs with canonical base pairing (hpWT), which were predominantly processed resulting in a loop.

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi.

Full-Length Hairpin RNA Accumulates at High Levels in Yeast but Not in Bacteria and Plants.

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria and were largely processed into shorter dsRNA fragments with no or few full-length molecules being present.

plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis.

DNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, ( () also known as ( and . While and were shown to function in disease response in somatic tissues, has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes.

Root-Specific Expression of a Jacalin Lectin Family Protein Gene Requires a Transposable Element Sequence in the Promoter.

Transposable elements (TEs) are widespread in the plant genome and can impact on the expression of neighbouring genes. Our previous studies have identified a number of DNA demethylase-regulated defence-related genes that contain TE sequences in the promoter and show tissue-specific expression in Arabidopsis. In this study we investigated the role of the promoter TE insertions in the root-specific expression of a DNA demethylase-regulated gene, , encoding a Jacalin lectin family protein.

Satellite RNA pathogens of plants: impacts and origins-an RNA silencing perspective.

Viral satellite RNAs (satRNAs) are among the smallest RNA pathogens in plants. They have little or no protein-coding capacity but can have a major impact on the host plants through trilateral interactions with helper viruses and host plants. Studies around the 1980s revealed much of what we know about satRNAs: they can affect helper virus accumulation, modulate helper virus-induced disease symptoms, and induce their own symptoms with the assistance of helper viruses which depend on specific nucleotide sequences of their genome and host species.

Satellite RNAs interfere with the function of viral RNA silencing suppressors.

Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N.

Cloning and profiling of small RNAs from cucumber mosaic virus satellite RNA.

RNA silencing is not only a gene regulation mechanism that is conserved in a broad range of eukaryotes but also an adaptive immune response against foreign nucleic acids including viruses in plants. A major feature of RNA silencing is the production of small RNA (sRNA) of 21-24 nucleotides (nt) in length from double-stranded (ds) or hairpin-like (hp) RNA by Dicer-like (DCL) proteins. These sRNAs guide the binding and cleavage of cognate single-stranded (ss) RNA by an RNA silencing complex.

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

Background: DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear.

In Nicotiana species, an artificial microRNA corresponding to the virulence modulating region of Potato spindle tuber viroid directs RNA silencing of a soluble inorganic pyrophosphatase gene and the development of abnormal phenotypes.

Potato spindle tuber viroid (PSTVd) is a small non-protein-coding RNA pathogen that can induce disease symptoms in a variety of plant species. How PSTVd induces disease symptoms is a long standing question. It has been suggested that PSTVd-derived small RNAs (sRNAs) could direct RNA silencing of a targeted host gene(s) resulting in symptom development.

Radiolabelling of steroids: the synthesis of 17α-[4-(14) C]trenbolone.

17β-Hydroxyestra-4,9,11-trien-3-one or trenbolone is an anabolic steroid used in some meat producing countries where its use is licenced. In cattle it is metabolised into 17α-trenbolone. We were required to make 17α-[4-(14) C]trenbolone for use in environmental fate studies.

Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum.

Background: Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain.

A fast and efficient method for preparation of high-quality RNA from fungal mycelia.

Background: Fungal RNA samples are usually isolated from fungal mycelia grown in liquid culture, which relies on prolific growth of the fungus in liquid media. The fungal biomass is then collected by vacuum filtration, which can result in low recovery for samples with reduced biomass due to poor growth in liquid media.

Findings: Here we report an alternative culturing method, based on growth on solid media which is independent of the ability of a fungus to grow in liquid culture.

Isolation and detection of small RNAs from plant tissues.

In plants, several classes of non-coding small RNA (sRNA) have been shown to be important regulators of gene expression in a wide variety of biological processes. The two main classes of sRNA, the small-interfering RNA (siRNA) and microRNA (miRNA) classes, are well documented and several experimental approaches have been developed to allow for their routine isolation and detection from plant tissues. Here, we describe the current methods used for the isolation of total RNA and the subsequent enrichment of low-molecular-weight (LMW) RNA species, as well as to outline how sRNAs are detected from such nucleic acid preparations.

RNA silencing and plant viral diseases.

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures.

Taxonomic revision and phylogenetic analysis of the flightless Mancallinae (Aves, Pan-Alcidae).

Although flightless alcids from the Miocene and Pliocene of the eastern Pacific Ocean have been known for over 100 years, there is no detailed evaluation of diversity and systematic placement of these taxa. This is the first combined analysis of morphological and molecular data to include all extant alcids, the recently extinct Great Auk Pinguinus impennis, the mancalline auks, and a large outgroup sampling of 29 additional non-alcid charadriiforms. Based on the systematic placement of Mancallinae outside of crown clade Alcidae, the clade name Pan-Alcidae is proposed to include all known alcids.

Viral small interfering RNAs target host genes to mediate disease symptoms in plants.

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI.

Transgene expression and transgene-induced silencing in diploid and autotetraploid Arabidopsis.

Previous studies have suggested that transgene expression in plants can be affected by ploidy. Here we show that three different transgenes, a reporter transgene, an antisense transgene, and a hairpin RNA (hpRNA) transgene, are all expressed at a lower level in autotetraploid (4n) than in diploid (2n) Arabidopsis. RNA silencing of two endogenous genes was induced by the antisense and hpRNA transgenes and this silencing is significantly less effective in 4n than in 2n Arabidopsis; furthermore, the reduced silencing in 4n Arabidopsis correlated with reduced accumulation of silencing-inducer RNAs.

Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana.

We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis.

The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs.

Viral small interfering RNA (siRNA) accumulation in plants is reported to exhibit a strong strand polarity bias, with plus (+) strand siRNAs dominating over minus (-) strand populations. This is of particular interest, as siRNAs processed from double-stranded RNA would be expected to accumulate equivalent amounts of both species. Here, we show that, as reported, (-) strand viral siRNAs are detected at much lower levels than (+) strand-derived species using standard Northern hybridization approaches.

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes.

In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs.

Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer.

RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3' terminal 2'-O-methyl group or was phosphorylated at the 5' terminus.