contributed equally to this work Temperature dramatically affects plant±virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus-and transgenederived small (21±26 nucleotide) interfering (si) RNAsÐthe central molecules of RNA silencing-mediated defence pathwaysÐare dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not in¯uence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.

MicroRNAs (miRNAs) are short, about 21 nucleotides in length, noncoding, regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Intensive miRNA research has necessitated the development of effective miRNA detection methods such as northern analyses, quantitative real-time PCR and microarrays. Northern analysis is a widely used method for miRNA analyses because it is generally a readily available technology for laboratories and does not require special equipment and technical knowledge. The major disadvantages of the northern blot technology using the traditional DNA oligonucleotide probes are its poor sensitivity and the high time consumption. Here, we describe an improved protocol for miRNA northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis and northern blotting, and the hybridization and detection of locked nucleic acid (LNA)-modified oligonucleotide probes. The use of LNA-modified oligonucleotide probes allows highly sensitive and specific detection of mature miRNAs and also dramatically reduces the period of time necessary for carrying out the protocol. Using this approach, the hybridization, washing and signal-detection steps can be performed ideally in 4 h.

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Although many subviral RNAs reduce or intensify disease symptoms caused by the helper virus, only recently have clues concerning the mechanism of disease modulation been revealed. New models for DI RNA-mediated reduction in helper virus levels and symptom attenuation include DI RNA enhancement of posttranscriptional gene silencing (PTGS), which is an antiviral defense mechanism in plants. Symptom enhancement by the satRNA of Cucumber mosaic virus is caused by minus-strand induction of the programmed cell death pathway. In contrast, symptom enhancement by satC of Turnip crinkle virus is due to satC interference with virion formation, leading to increased levels of free coat protein, which is the viral suppressor of PTGS. Mutualism between satRNA and helper virus can be seen for the satRNA of Groundnut rosette virus, which contributes to the virus by allowing virion assembly. These novel findings are leading to re-evaluation of the relationships between subviral RNAs, helper viruses, and hosts.

Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus-infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus-occupied sectors, is mediated mainly by the Tombusvirus p19 RNA-silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down-regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus-infected plants, indicating that this activity is uncoupled from the small RNA-binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti-viral function of AGO1 protein.

SummaryMicroRNAs (miRNAs) are an abundant class of small, endogenous non-protein-coding RNAs, approximately 21 nucleotides in length, that modulate the expression of animal and plant target genes at the posttranscriptional level. Recent work has shown that miRNA-based gene regulation plays a crucial role in pathways involved in plant growth and development. However, knowledge about the timing and spatial regulation of plant miRNA expression is still limited. Here we used in situ analysis to demonstrate that miRNAs accumulate spatially and temporally in a highly restricted manner in Nicotiana benthamiana and Arabidopsis thaliana. The presence of the seven investigated miRNAs was characteristic of the developing organs, implying a role in cell-fate establishment, differentiation and cell-cycle progression. Spatial analyses revealed that six of the studied miRNAs were present in vascular bundles, suggesting that mobile miRNAs in the phloem could contribute to the coordination of organogenesis and development. The obvious absence of miR167 in vascular bundles represented an exception to this observation, implying an active process in regulating the presence of miRNAs in the vascular system. Taken together, our results imply that the spatially and temporally organized accumulation of miRNAs plays a pivotal role in fine-tuning of target gene expression in plant development.

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus-and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.RNA silencing (also termed posttranscriptional gene silencing in plants and RNA interference in animals) is a conserved eukaryotic gene inactivation system that plays regulatory roles in many biological processes including development, maintenance of genome stability, and antiviral responses (2,6,12,25,54). RNA silencing is induced by accumulation of doublestranded RNAs (dsRNAs). dsRNAs are first processed by an RNase III-like nuclease called DICER (in plants termed DICER-LIKE, or DCL) into short (21 to 25 nucleotide [nt]) RNAs, and then these short RNAs incorporate and guide different silencing effector complexes to homologous nucleic acids for suppression (2,6,12,16,25,54). In plants, RNA silencing acts at both single-cell (cell-autonomous silencing) and at whole-plant (systemic silencing) levels. Cell-autonomous silencing inactivates genes in the cells in which dsRNAs accumulated. Moreover, cell-autonomous silencing generates mobile silencing signals that confer suppression of homologous mRNAs in neighboring cells (short distance) and in distant tissues (long-distance systemic silencing) (29,31,32,56).DICERs can process dsRNAs into two functionally different small RNAs, micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are involved in the control of many endogenous protein-encoding mRNAs...

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.