Genome-wide identification of gene families related to miRNA biogenesis in Mangifera indica L. and their possible role during heat stress.

Mango is a popular tropical fruit that requires quarantine hot water treatment (QHWT) for postharvest sanitation, which can cause abiotic stress. Plants have various defense mechanisms to cope with stress; miRNAs mainly regulate the expression of these defense responses. Proteins involved in the biogenesis of miRNAs include DICER-like (DCL), ARGONAUTE (AGO), HYPONASTIC LEAVES 1 (HYL1), SERRATE (SE), HUA ENHANCER1 (HEN1), HASTY (HST), and HEAT-SHOCK PROTEIN 90 (HSP90), among others. According to our analysis, the mango genome contains five DCL, thirteen AGO, six HYL, two SE, one HEN1, one HST, and five putative HSP90 genes. Gene structure prediction and domain identification indicate that sequences contain key domains for their respective gene families, including the RNase III domain in DCL and PAZ and PIWI domains for AGOs. In addition, phylogenetic analysis indicates the formation of clades that include the mango sequences and their respective orthologs in other flowering plant species, supporting the idea these are functional orthologs. The analysis of cis-regulatory elements of these genes allowed the identification of MYB, ABRE, GARE, MYC, and MeJA-responsive elements involved in stress responses. Gene expression analysis showed that most genes are induced between 3 to 6 h after QHWT, supporting the early role of miRNAs in stress response. Interestingly, our results suggest that mango rapidly induces the production of miRNAs after heat stress. This research will enable us to investigate further the regulation of gene expression and its effects on commercially cultivated fruits, such as mango, while maintaining sanitary standards.

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling.

Small RNA (sRNA)-mediated RNA silencing (also known as RNA interference, or RNAi) is a conserved mechanism in eukaryotes that includes RNA degradation, DNA methylation, heterochromatin formation and protein translation repression. In plants, sRNAs can move either cell-to-cell or systemically, thereby acting as mobile silencing signals to trigger noncell autonomous silencing. However, whether and what proteins are also involved in noncell autonomous silencing have not been elucidated. In this study, we utilized a previously reported inducible RNAi plant, PDSi, which can induce systemic silencing of the endogenous PDS gene, and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity. We confirmed that the C-terminus of DCL3, including the predicted RNA-binding domain, is capable of binding short RNAs. Mutations affecting RNA binding, but not processing activity, reduced systemic PDS silencing, indicating that DCL3 binding to RNAs is required for the induction of systemic silencing. Cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves. Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00124-6.

Chemical shift assignment of dsRBD1 and dsRBD2 of Arabidopsis thaliana DRB3, an essential protein involved in RNAi-mediated antiviral defense.

As sessile organisms, plants need to counteract different biotic and abiotic stresses to survive. RNA interference provides natural immunity against various plant pathogens, especially against viral infections via inhibition of viral genome replication or translation. In plants, DRB3, a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD), plays a vital role in RNA-directed DNA methylation of the geminiviral genome. Additionally, DRB3 arrests the replication of the viral genome in the viral replication complex of RNA viruses through a mechanism that has yet to be fully deciphered. Therefore, as a first step towards exploring the structural details of DRB3, we present a nearly complete backbone and side chain assignment of the two N-terminal dsRBD domains.

Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

Genome-wide identification of DCL, AGO, and RDR gene families in wheat (Triticum aestivum L.) and their expression analysis in response to heat stress.

Key components of the RNA interference (RNAi) pathway include the Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. While these components have been studied in various plant species, their functional validation in wheat remains unexplored particularly under heat stress. In this study, a comprehensive genome-wide analysis to identify, and characterize DCL, AGO, and RDR genes in wheat and their expression patterns was carried out. Using phylogenetic analysis with orthologous genes from Arabidopsis and rice, we identified a total of 82 AGO, 31 DCL, and 31 RDR genes distributed across the 21 chromosomes of wheat. To understand the regulatory network, a network analysis of miRNAs that target RNA-silencing genes was performed. Our analysis revealed that 13 miRNAs target AGO genes, 8 miRNAs target DCL genes, and 10 miRNAs target RDR genes at different sites, respectively. Additionally, promoter analysis of the RNA-silencing genes was done and identified the presence of 132 cis-elements responsive to stress and phytohormones. To examine their expression patterns, we performed RNA-seq analysis in the flag leaf samples of wheat exposed to both normal and heat stress conditions. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to gradient heat stress treatments. Our results showed constitutive expression of the AGO1, AGO9, and DCL2 gene families, indicating their importance in the overall biological processes of wheat. Notably, RDR1, known to be involved in small interfering RNA (siRNA) biogenesis, exhibited higher expression levels in wheat leaf tissues. These findings suggest that these genes may play a role in responses to stress in wheat, highlighting their significance in adapting to environmental challenges. Overall, our study provides additional knowledge to understand the mechanisms underlying heat stress responses and emphasizes the essential roles of these gene families in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01362-0.

Bacillus subtilis 26D Triggers Induced Systemic Resistance against Rhopalosiphum padi L. by Regulating the Expression of Genes AGO, DCL and microRNA in Bread Spring Wheat.

Bacillus subtilis 26D is a plant growth-promoting endophytic bacteria capable of inducing systemic resistance through the priming mechanism, which includes plant genome reprogramming and the phenomenon of RNA interference (RNAi) and microRNA (miRNAs). The phloem-feeding insect bird cherry-oat aphid Rhopalosiphum padi L. is a serious pest that causes significant damage to crops throughout the world. However, the function of plant miRNAs in the response to aphid infestation remains unclear. The results of this work showed that B. subtilis 26D stimulated aphid resistance in wheat plants, inducing the expression of genes of hormonal signaling pathways ICS, WRKY13, PR1, ACS, EIN3, PR3, and ABI5. In addition, B. subtilis 26D activated the RNAi mechanism and regulated the expression of nine conserved miRNAs through activation of the ethylene, salicylic acid (SA), and abscisic acid (ABA) signaling pathways, which was demonstrated by using treatments with phytohormones. Treatment of plants with SA, ethylene, and ABA acted in a similar manner to B. subtilis 26D on induction of the expression of the AGO4, AGO5 and DCL2, DCL4 genes, as well as the expression of nine conserved miRNAs. Different patterns of miRNA expression were found in aphid-infested plants and in plants treated with B. subtilis 26D or SA, ethylene, and ABA and infested by aphids, suggesting that miRNAs play multiple roles in the plant response to phloem-feeding insects, associated with effects on hormonal signaling pathways, redox metabolism, and the synthesis of secondary metabolites. Our study provides new data to further elucidate the fine mechanisms of bacterial-induced priming. However, further extensive work is needed to fully unravel these mechanisms.

DICER-LIKE2 Plays a Crucial Role in Rice Stripe Virus Coat Protein-Mediated Virus Resistance in Arabidopsis.

Virus coat protein (CP)-mediated resistance is considered an effective antiviral defense strategy that has been used to develop robust resistance to viral infection. Rice stripe virus (RSV) causes significant losses in rice production in eastern Asia. We previously showed that the overexpression of RSV CP in Arabidopsis plants results in immunity to RSV infection, using the RSV-Arabidopsis pathosystem, and this CP-mediated viral resistance depends on the function of DCLs and is mostly involved in RNA silencing. However, the special role of DCLs in producing t-siRNAs in CP transgenic Arabidopsis plants is not fully understood. In this study, we show that RSV CP transgenic Arabidopsis plants with the dcl2 mutant background exhibited similar virus susceptibility to non-transgenic plants and were accompanied by the absence of transgene-derived small interfering RNAs (t-siRNAs) from the CP region. The dcl2 mutation eliminated the accumulation of CP-derived t-siRNAs, including those generated by other DCL enzymes. In contrast, we also developed RSV CP transgenic Arabidopsis plants with the dcl4 mutant background, and these CP transgenic plants showed immunity to virus infection and accumulated comparable amounts of CP-derived t-siRNAs to CP transgenic Arabidopsis plants with the wild-type background except for a significant increase in the abundance of 22 nt t-siRNA reads. Overall, our data indicate that DCL2 plays an essential, as opposed to redundant, role in CP-derived t-siRNA production and induces virus resistance in RSV CP transgenic Arabidopsis plants.

Identification and Analysis of Protein Family Associated with RNA Interference Pathway in Juglandaceae.

BACKGROUND: One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits. METHODS: In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut (Juglans rega) genome. RESULTS: 5 DCL genes, 13 RDR genes, 15 DRB genes, and 15 AGO genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression. CONCLUSIONS: In this study, the genome of the Juglandaceae's DCL, RDR, DRB, and AGO gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.

Synthesis, 123I-Radiolabeling Optimization, and Initial Preclinical Evaluation of Novel Urea-Based PSMA Inhibitors with a Tributylstannyl Prosthetic Group in Their Structures.

Prostate-specific membrane antigen (PSMA) has been identified as a target for the development of theranostic agents. In our current work, we describe the design and synthesis of novel N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-L-lysine (DCL) urea-based PSMA inhibitors with a chlorine-substituted aromatic fragment at the lysine ε-nitrogen atom, a dipeptide including two phenylalanine residues in the L-configuration as the peptide fragment of the linker, and 3- or 4-(tributylstannyl)benzoic acid as a prosthetic group in their structures for radiolabeling. The standard compounds [127I]PSMA-m-IB and [127I]PSMA-p-IB for comparative and characterization studies were first synthesized using two alternative synthetic approaches. An important advantage of the alternative synthetic approach, in which the prosthetic group (NHS-activated esters of compounds) is first conjugated with the polypeptide sequence followed by replacement of the Sn(Bu)3 group with radioiodine, is that the radionuclide is introduced in the final step of synthesis, thereby minimizing operating time with iodine-123 during the radiolabeling process. The obtained DCL urea-based PSMA inhibitors were radiolabeled with iodine-123. The radiolabeling optimization results showed that the radiochemical yield of [123I]PSMA-p-IB was higher than that of [123I]PSMA-m-IB, which were 74.9 ± 1.0% and 49.4 ± 1.2%, respectively. The radiochemical purity of [123I]PSMA-p-IB after purification was greater than 99.50%. The initial preclinical evaluation of [123I]PSMA-p-IB demonstrated a considerable affinity and specific binding to PC-3 PIP (PSMA-expressing cells) in vitro. The in vivo biodistribution of this new radioligand [123I]PSMA-p-IB showed less accumulation than [177Lu]Lu-PSMA-617 in several normal organs (liver, kidney, and bone). These results warrant further preclinical development, including toxicology evaluation and experiments in tumor-bearing mice.

Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context.

BACKGROUND: In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. METHODS: In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts. RESULTS: DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci. CONCLUSIONS: We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.

Use of liposome-encapsulated estetrol for treatment of neonatal hypoxic-ischemic encephalopathy.

Estetrol (E4) is a natural estrogen synthesized only during pregnancy. It has strong neuroprotective and antioxidative activities. The aim of the present study was to define the neuroprotective potency of E4 encapsulated either in liposome (Lipo-E4) or in drug-in cyclodextrin (HP-ß-CD) in liposome (DCL) system, and compare them with a single use of E4. In vitro studies were performed in an oxidative stress model of primary hippocampal neuronal cell cultures, followed by the lactate dehydrogenase activity and cell proliferation assays. In vivo studies were conducted by using a model of neonatal hypoxic-ischemic encephalopathy in immature rat pups. Brain samples were studied by (immuno)histochemistry for the detection of survived cells, expression of microtubule-associated protein-2, myelin basic protein, doublecortin and vascular-endothelial growth factor. Concentrations of glial fibrillary acidic protein in blood serum were studied by ELISA. In vitro, cell proliferation was significantly up-regulated in cultures treated either by DCL-E4 or E4 compared to the control cells, whereas DCL-E4 treated cells had significantly higher survival rate than the cells treated by E4 alone. Evaluation of brain samples showed that DCL-E4 and a high dose of E4 alone significantly preserve the grey and the white matter loses, and diminish GFAP expression in blood. Although DCL-E4 and E4 have similar effect on neurogenesis in the hippocampus and the cortex, DCL-E4 treatment significantly up-regulates angiogenesis in the hippocampus compared to a single use of E4. Present work reveals for the first time that liposome-encapsulated E4 might be a better alternative to a single use of E4.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app.

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Small Non-coding RNA in Plants: From Basic Science to Innovative Applications.

Plants possess an arsenal of different classes of small RNAs (sRNAs) of variable size, which play a regulatory role in a multitude of physiological and pathological processes via transcriptional or post-transcriptional gene silencing. The hard challenges that agriculture will face in the next few decades, such as an increasing demand for agrifood production related to the global increase in population, have stimulated the development of innovative biotechnological approaches in agriculture. In this regard, the use of artificial sRNAs has already been exploited successfully for many purposes, including control of severe plant diseases, improvement of genetic and agronomic traits of cultivated species, and increasing the nutritional value of plant foodstuffs. This strategy relies on the application of synthetic sRNA molecules to induce specific physiological responses by triggering appropriate RNA silencing pathways. This review contextualizes the use of artificial sRNAs in consideration of the huge diversity of RNA silencing mechanisms in plants. Additionally, the discussion also examines microRNAs from edible plants and exosome-like vesicles, also known as plant-derived edible nanoparticles (ENPs), which themselves can act as micronutrients.

Genome-wide analysis of key gene families in RNA silencing and their responses to biotic and drought stresses in adzuki bean.

BACKGROUND: In plants, RNA silencing is an important conserved mechanism to regulate gene expression and combat against abiotic and biotic stresses. Dicer-like (DCL) and Argonaute (AGO) proteins and RNA-dependent RNA polymerase (RDR) are the core elements involved in gene silencing and their gene families have been explored in many plants. However, these genes and their responses to stresses have not yet been well characterized in adzuki bean. RESULTS: A total of 11 AGO, 7 DCL and 6 RDR proteins were identified, and phylogenetic analyses of these proteins showed that they clustered into six, four and four clades respectively. The expression patterns of these genes in susceptible or resistant adzuki bean cultivars challenged with drought, bean common mosaic virus and Podosphaera xanthii infections were further validated by quantitative RT-PCR. The different responses of these proteins under abiotic and biotic stresses indicated their specialized regulatory mechanisms. CONCLUSIONS: In this study, 24 genes of the DCL, AGO and RDR gene families in adzuki bean were identified, and the sequence characterization, structure of the encoded proteins, evolutionary relationship with orthologues in other legumes and gene expression patterns under drought and biotic stresses were primarily explored, which enriched our understanding of these genes in adzuki bean. Our findings provide a foundation for the comparative genomic analyses of RNA silencing elements in legume plants and further new insights into the functional complexity of RNA silencing in the response to various stresses in adzuki bean.

microRNA production in Arabidopsis.

In plants, microRNAs (miRNAs) associate with ARGONAUTE (AGO) proteins and act as sequence-specific repressors of target gene expression, at the post-transcriptional level through target transcript cleavage and/or translational inhibition. MiRNAs are mainly transcribed by DNA-dependent RNA polymerase II (POL II) and processed by DICER LIKE1 (DCL1) complex into 21∼22 nucleotide (nt) long. Although the main molecular framework of miRNA biogenesis and modes of action have been established, there are still new requirements continually emerging in the recent years. The studies on the involvement factors in miRNA biogenesis indicate that miRNA biogenesis is not accomplished separately step by step, but is closely linked and dynamically regulated with each other. In this article, we will summarize the current knowledge on miRNA biogenesis, including MIR gene transcription, primary miRNA (pri-miRNA) processing, miRNA AGO1 loading and nuclear export; and miRNA metabolism including methylation, uridylation and turnover. We will describe how miRNAs are produced and how the different steps are regulated. We hope to raise awareness that the linkage between different steps and the subcellular regulation are becoming important for the understanding of plant miRNA biogenesis and modes of action.

CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.

Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.

Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing.

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER-LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation in vivo and in vitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupting SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purification of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing.

Genome-Wide Identification and Characterization of Argonaute, Dicer-like and RNA-Dependent RNA Polymerase Gene Families and Their Expression Analyses in Fragaria spp.

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.

Keeping up with the miRNAs: current paradigms of the biogenesis pathway.

For many years we have studied the processes involved in producing miRNAs in plants and the numerous differences from their metazoan counterpart. A well-defined catalytic process, mostly carried out by the RNase III enzyme DICER-LIKE1 (DCL1), it was identified early after the discovery of RNAi and was followed by the isolation of a plethora of miRNA biogenesis cofactors. The production of miRNAs, which later are loaded in ARGONAUTE (AGO) proteins to perform their RNA silencing functions both within the cell and non-cell autonomously, appears to be a highly regulated and dynamic process. Many regulatory events during miRNA biogenesis require the action of specific proteins. However, in recent years, many post-transcriptional modifications, structural features, and coupling with other cellular processing emerged as critical elements controlling the production of miRNA and, thus, a plant's physiology. This review discusses new evidence that has changed the way we understand how miRNAs are produced in plants. We also provide an updated view of the miRNA biogenesis pathways, focusing on the gaps in our knowledge and the most compelling questions that remain open.

The dicing activity of DCL3 and DCL4 is negatively affected by flavonoids.

KEY MESSAGE: The dicing activities of DCL3 and DCL4 are inhibited by accumulated metabolites in soybean leaves. Epicatechin and 7,4'-dihydroxyflavone inhibited Arabidopsis DCL3 and DCL4 in vitro. Flavonoids are major secondary metabolites in plants, and soybean (Glycine max L.) is a representative plant that accumulates flavonoids, including isoflavonoids, to high levels. Naturally-occurring RNA interference (RNAi) against the chalcone synthase (CHS) gene represses flavonoid (anthocyanin) biosynthesis in an organ-specific manner, resulting in a colorless (yellow) seed coat in many soybean cultivars. To better understand seed coat-specific naturally-occurring RNAi in soybean, we characterized soybean Dicer-like (DCL) 3 and 4, which play critical roles in RNAi. Using a previously established dicing assay, two dicing activities producing 24- and 21-nt siRNAs, corresponding to DCL3 and DCL4, respectively, were detected in soybean. Dicing activity was detected in colorless seed coats where RNAi against CHS genes was found, but no dicing activity was detected in leaves where CHS expression was prevalent. Biochemical analysis revealed that soybean leaves contained two types of inhibitors effective for Arabidopsis Dicers (AtDCL3 and AtDCL4), one of which was a heat-labile high molecular weight compound of 50 to 100 kD while another was a low molecular weight substance. We found that some flavonoids, such as epicatechin and 7,4'-dihydroxyflavone, inhibited both AtDCL3 and AtDCL4, but AtDCL4 was more sensitive to these flavonoids than AtDCL3. These results suggest that flavonoids inhibit the dicing activity of DCL4 and thereby attenuate RNAi in soybean leaves.