24-nt siRNAs Research Articles

Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection.

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

Virus-Mediated Targeted DNA Methylation Illuminates the Dynamics of Methylation in an Endogenous Plant Gene.

DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3′ end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.

Genome-wide analysis of long noncoding RNAs, 24-nt siRNAs, DNA methylation and H3K27me3 marks in Brassica rapa.

Long noncoding RNAs (lncRNAs) are RNA fragments that generally do not code for a protein but are involved in epigenetic gene regulation. In this study, lncRNAs of Brassica rapa were classified into long intergenic noncoding RNAs, natural antisense RNAs, and intronic noncoding RNAs and their expression analyzed in relation to genome-wide 24-nt small interfering RNAs (siRNAs), DNA methylation, and histone H3 lysine 27 trimethylation marks (H3K27me3). More than 65% of the lncRNAs analyzed consisted of one exon, and more than 55% overlapped with inverted repeat regions (IRRs). Overlap of lncRNAs with IRRs or genomic regions encoding for 24-nt siRNAs resulted in increased DNA methylation levels when both were present. LncRNA did not overlap greatly with H3K27me3 marks, but the expression level of intronic noncoding RNAs that did coincide with H3K27me3 marks was higher than without H3K27me3 marks. The Brassica genus comprises important vegetables and oil seed crops grown across the world. B. rapa is a diploid (AA genome) thought to be one of the ancestral species of both B. juncea (AABB genome) and B. napus (AACC) through genome merging (allotetrapolyploidization). Complex genome restructuring and epigenetic alterations are thought to be involved in these allotetrapolyploidization events. Comparison of lncRNAs between B. rapa and B. nigra, B. oleracea, B. juncea, and B. napus showed the highest conservation with B. oleracea. This study presents a comprehensive analysis of the epigenome structure of B. rapa at multi-epigenetic levels (siRNAs, DNA methylation, H3K27me3, and lncRNAs) and identified a suite of candidate lncRNAs that may be epigenetically regulated in the Brassica genus.

On the Role of Transposable Elements in the Regulation of Gene Expression and Subgenomic Interactions in Crop Genomes

Transposable elements (TEs) represent a major and variable portion of plant genomes, and recent progress in genetics and genomics has highlighted the importance of different TE species as a useful genetic tool in crop breeding. TEs can cause changes in the pattern of gene expression, and regulate gene function by various means such as cis- up- or down-regulation of nearby genes through insertion at promoter, intron, exon and down-stream regions, and trans-production of short interfering RNAs (siRNAs) via two RNA-directed DNA methylation (RdDM) pathways. siRNAs generated through different RdDM pathways differ in length and have variable effects on TEs. For instance, noncoding siRNAs of 20–60 nt produced by RNA polymerase IV (dicer-independent) and 21/22 nt by Pol II (dicer-dependent) have only minor effects on TEs compared with 24 nt siRNAs produced by Pol IV (dicer-dependent pathways). Following whole-genome duplication (WGD) events after polyploidization in allopolyploids, TEs from either parent are able to induce siRNAs to regulate the complex polyploid genome. Those designated as ‘controllers’ usually reside in the dominant parent and affect the TEs of the recessive parent. Subgenome cross-talk thus appears to contribute to epigenetic regulation as well as reshuffling or restructuring of subgenomes and creation of novel patterns of genes expression/and variation in local or global copy number. In this review, we focus on recent progress in unraveling the role of TEs in gene expression regulation via TE-derived siRNAs in the context of polyploid plant evolution and environmental stress, and explore how ancient WGD and recent polyploidy affected the evolution of TE-induced epigenetic mechanisms.

Ribosome stalling and SGS3 phase separation prime the epigenetic silencing of transposons.

Transposable elements (TEs, transposons) are mobile DNAs that can cause fatal mutations1. To suppress their activity, host genomes deploy small interfering RNAs (siRNAs) that trigger and maintain their epigenetic silencing2,3. Whereas 24-nucleotide (nt) siRNAs mediate RNA-directed DNA methylation (RdDM) to reinforce the silent state of TEs3, activated or naive TEs give rise to 21- or 22-nt siRNAs by the RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)-mediated pathway, triggering both RNAi and de novo DNA methylation4,5. This process, which is called RDR6-RdDM, is critical for the initiation of epigenetic silencing of active TEs; however, their specific recognition and the selective processing of siRNAs remain elusive. Here, we suggest that plant transposon RNAs undergo frequent ribosome stalling caused by their unfavourable codon usage. Ribosome stalling subsequently induces RNA truncation and localization to cytoplasmic siRNA bodies, both of which are essential prerequisites for RDR6 targeting6,7. In addition, SUPPRESSOR OF GENE SILENCING 3 (SGS3), the RDR6-interacting protein7, exhibits phase separation both in vitro and in vivo through its prion-like domains, implicating the role of liquid-liquid phase separation in siRNA body formation. Our study provides insight into the host recognition of active TEs, which is important for the maintenance of genome integrity.

Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

BackgroundSmall RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action.ResultsWe investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy.ConclusionsMBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

The diversity of post-transcriptional gene silencing mediated by small silencing RNAs in plants.

In plants, post-transcriptional gene silencing (PTGS) tightly regulates development, maintains genome stability and protects plant against foreign genes. PTGS can be triggered by virus infection, transgene, and endogenous transcript, thus commonly serves as an RNA-based immune mechanism. Accordingly, based on the initiating factors, PTGS can be divided into viral-PTGS, transgene-PTGS, and endo-gene-PTGS. Unlike the intensely expressed invading transgenes and viral genes that frequently undergo PTGS, most endogenous genes do not trigger PTGS, except for a few that can produce endogenous small RNAs (sRNAs), including microRNA (miRNA) and small interfering RNA (siRNA). Different lengths of miRNA and siRNA, mainly 21-, 22- or 24-nucleotides (nt) exert diverse functions, ranging from target mRNA degradation, translational inhibition, or DNA methylation and chromatin modifications. The abundant 21-nt miRNA or siRNA, processed by RNase-III enzyme DICER-LIKE 1 (DCL1) and DCL4, respectively, have been well studied in the PTGS pathways. By contrast, the scarceness of endogenous 22-nt sRNAs that are primarily processed by DCL2 limits their research, although a few encouraging studies have been reported recently. Therefore, we review here our current understanding of diverse PTGS pathways triggered by a variety of sRNAs and summarize the distinct features of the 22-nt sRNA mediated PTGS.

Gene capture by transposable elements leads to epigenetic conflict in maize

Transposable elements (TEs) regularly capture fragments of genes. When the host silences these TEs, siRNAs homologous to the captured regions may also target the genes. This epigenetic crosstalk establishes an intragenomic conflict: silencing the TEs has the cost of silencing the genes. If genes are important, however, natural selection may maintain function by moderating the silencing response, which may also advantage the TEs. In this study, we examined this model by focusing on Helitrons, Pack-MULEs, and Sirevirus LTR retrotransposons in the maize genome. We documented 1263 TEs containing exon fragments from 1629 donor genes. Consistent with epigenetic conflict, donor genes mapped more siRNAs and were more methylated than genes with no evidence of capture. However, these patterns differed between syntelog versus translocated donor genes. Syntelogs appeared to maintain function, as measured by gene expression, consistent with moderation of silencing for functionally important genes. Epigenetic marks did not spread beyond their captured regions and 24nt crosstalk siRNAs were linked with CHH methylation. Translocated genes, in contrast, bore the signature of silencing. They were highly methylated and less expressed, but also overrepresented among donor genes and located away from chromosomal arms, which suggests a link between capture and gene movement. Splitting genes into potential functional categories based on evolutionary constraint supported the synteny-based findings. TE families captured genes in different ways, but the evidence for their advantage was generally less obvious; nevertheless, TEs with captured fragments were older, mapped fewer siRNAs, and were slightly less methylated than TEs without captured fragments. Collectively, our results argue that TE capture triggers an intragenomic conflict that may not affect the function of important genes but may lead to the pseudogenization of less-constrained genes.

Chromatin regulates expression of small RNAs to help maintain transposon methylome homeostasis in Arabidopsis

BackgroundEukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation, but little is known about how chromatin regulates siRNA expression.ResultsWe demonstrate that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. TEs generate siRNAs throughout embryogenesis according to two distinct patterns depending on whether they are located in euchromatic or heterochromatic regions of the genome. siRNA precursors are transcribed in embryos, and siRNAs are required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states.ConclusionsDecondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to help reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.

Abundant expression of maternal siRNAs is a conserved feature of seed development

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

Plant 22-nt siRNAs mediate translational repression and stress adaptation.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases-NIA1 and NIA2-produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.

Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants.

Plant endogenous small RNAs (sRNAs) are important regulators of gene expression. There are two broad categories of plant sRNAs: microRNAs (miRNAs) and endogenous short interfering RNAs (siRNAs). MicroRNA loci are relatively well-annotated but compose only a small minority of the total sRNA pool; siRNA locus annotations have lagged far behind. Here, we used a large data set of published and newly generated sRNA sequencing data (1333 sRNA-seq libraries containing more than 20 billion reads) and a uniform bioinformatic pipeline to produce comprehensive sRNA locus annotations of 47 diverse plants, yielding more than 2.7 million sRNA loci. The two most numerous classes of siRNA loci produced mainly 24- and 21-nucleotide (nt) siRNAs, respectively. Most often, 24-nt-dominated siRNA loci occurred in intergenic regions, especially at the 5′-flanking regions of protein-coding genes. In contrast, 21-nt-dominated siRNA loci were most often derived from double-stranded RNA precursors copied from spliced mRNAs. Genic 21-nt-dominated loci were especially common from disease resistance genes, including from a large number of monocots. Individual siRNA sequences of all types showed very little conservation across species, whereas mature miRNAs were more likely to be conserved. We developed a web server where our data and several search and analysis tools are freely accessible.

Arabidopsis RNA Polymerase IV generates 21-22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes.

The plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements (TEs), with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to TEs to reinforce the boundary between heterochromatin and euchromatin. In the male gametophytic phase of the plant life cycle, pollen, Pol IV switches to generating primarily 21-22 nt siRNAs, but the biogenesis and function of these siRNAs have been enigmatic. In contrast to being pollen-specific, we identified that Pol IV generates these 21-22 nt siRNAs in sporophytic tissues, likely from the same transcripts that are processed into the more abundant 24 nt siRNAs. The 21-22 nt forms are specifically generated by the combined activities of DICER proteins DCL2/DCL4 and can participate in RNA-directed DNA methylation. These 21-22 nt siRNAs are also loaded into ARGONAUTE1 (AGO1), which is known to function in post-transcriptional gene regulation. Like other plant siRNAs and microRNAs incorporated into AGO1, we find a signature of genic mRNA cleavage at the predicted target site of these siRNAs, suggesting that Pol IV-generated 21-22 nt siRNAs may function to regulate gene transcript abundance. Our data provide support for the existing model that in pollen Pol IV functions in gene regulation. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Construction and characterization of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare).

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Genome‐wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea

SummaryHybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24‐nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid‐MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down‐regulated hybrid‐MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Genome-wide redistribution of 24-nt siRNAs in rice gametes.

Gametes constitute a critical stage of the plant life cycle during which the genome undergoes reprogramming in preparation for embryogenesis. Here, we examined genome-wide distributions of small RNAs in the sperm and egg cells of rice. We found that 24-nt siRNAs, which are a hallmark of RNA-directed DNA methylation (RdDM) in plants, were depleted from heterochromatin boundaries in both gametes relative to vegetative tissues, reminiscent of siRNA patterns in DDM1-type nucleosome remodeler mutants. In sperm cells, 24-nt siRNAs were spread across heterochromatic regions, while in egg cells, 24-nt siRNAs were concentrated at a smaller number of heterochromatic loci throughout the genome, especially at loci which also produced siRNAs in other tissues. In both gametes, patterns of CHH methylation, typically a strong indicator of RdDM, were similar to vegetative tissues, although lower in magnitude. These findings indicate that the small RNA transcriptome undergoes large-scale redistribution in both male and female gametes, which is not correlated with recruitment of DNA methyltransferases in gametes and suggestive of unexplored regulatory activities of gamete small RNAs.

Small noncoding RNA discovery and profiling with sRNAtools based on high-throughput sequencing.

Small noncoding RNAs (sRNA/sncRNAs) are generated from different genomic loci and play important roles in biological processes, such as cell proliferation and the regulation of gene expression. Next-generation sequencing (NGS) has provided an unprecedented opportunity to discover and quantify diverse kinds of sncRNA, such as tRFs (tRNA-derived small RNA fragments), phasiRNAs (phased, secondary, small-interfering RNAs), Piwi-interacting RNA (piRNAs) and plant-specific 24-nt short interfering RNAs (siRNAs). However, currently available web-based tools do not provide approaches to comprehensively analyze all of these diverse sncRNAs. This study presents a novel integrated platform, sRNAtools (https://bioinformatics.caf.ac.cn/sRNAtools), that can be used in conjunction with high-throughput sequencing to identify and functionally annotate sncRNAs, including profiling microRNAss, piRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and rRNAs and discovering isomiRs, tRFs, phasiRNAs and plant-specific 24-nt siRNAs for up to 21 model organisms. Different modules, including single case, batch case, group case and target case, are developed to provide users with flexible ways of studying sncRNA. In addition, sRNAtools supports different ways of uploading small RNA sequencing data in a very interactive queue system, while local versions based on the program package/Docker/virtureBox are also available. We believe that sRNAtools will greatly benefit the scientific community as an integrated tool for studying sncRNAs.

WPMIAS: Whole-degradome-based Plant MicroRNA-Target Interaction Analysis Server.

It can be freely accessed at https://cbi.njau.edu.cn/WPMIAS/. Supplementary data are available at Bioinformatics online.

SiRNAs Derived from Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus Down-modulated the Expression Levels of Endogenous Genes in Phalaenopsis equestris.

Interplay between Cymbidium mosaic virus (CymMV)/Odontoglossum ringspot virus (ORSV) and its host plant Phalaenopsis equestris remain largely unknown, which led to deficiency of effective measures to control disease of P. equestris caused by infecting viruses. In this study, for the first time, we characterized viral small interfering RNAs (vsiRNAs) profiles in P. equestris co-infected with CymMV and ORSV through small RNA sequencing technology. CymMV and ORSV small interfering RNAs (siRNAs) demonstrated several general and specific/new characteristics. vsiRNAs, with A/U bias at the first nucleotide, were predominantly 21-nt long and they were derived predominantly (90%) from viral positive-strand RNA. 21-nt siRNA duplexes with 0-nt overhangs were the most abundant 21-nt duplexes, followed by 2-nt overhangs and then 1-nt overhangs 21-nt duplexes in infected P. equestris. Continuous but heterogeneous distribution and secondary structures prediction implied that vsiRNAs originate predominantly by direct Dicer-like enzymes cleavage of imperfect duplexes in the most folded regions of the positive strand of both viruses RNA molecular. Furthermore, we totally predicted 54 target genes by vsiRNAs with psRNATarget server, including disease/stress response–related genes, RNA interference core components, cytoskeleton-related genes, photosynthesis or energy supply related genes. Gene Ontology classification showed that a majority of the predicted targets were related to cellular components and cellular processes and performed a certain function. All target genes were down-regulated with different degree by vsiRNAs as shown by real-time reverse transcription polymerase chain reaction. Taken together, CymMV and ORSV siRNAs played important roles in interplay with P. equestris by down modulating the expression levels of endogenous genes in host plant.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

BackgroundMethylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert.ResultsWe show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases.ConclusionsOur results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.