An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length.

The plant siRNA landscape.

Whereas micro (mi)RNAs are considered the clean, noble side of the small RNA world, small interfering (si)RNAs are often seen as a noisy set of molecules whose barbarian acronyms reflect a large diversity of often elusive origins and functions. Twenty-five years after their discovery in plants, however, new classes of siRNAs are still being identified, sometimes in discrete tissues or at particular developmental stages, making the plant siRNA world substantially more complex and subtle than originally anticipated. Focusing primarily on the model Arabidopsis, we review here the plant siRNA landscape, including transposable elements (TE)-derived siRNAs, a vast array of non-TE-derived endogenous siRNAs, as well as exogenous siRNAs produced in response to invading nucleic acids such as viruses or transgenes.

In planta dynamics, transport biases, and endogenous functions of mobile siRNAs in Arabidopsis.

In RNA interference (RNAi), small interfering RNAs (siRNAs) produced from double-stranded RNA guide ARGONAUTE (AGO) proteins to silence sequence-complementary RNA/DNA. RNAi can propagate locally and systemically in plants, but despite recent advances in our understanding of the underlying mechanisms, basic questions remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata (PDs), yet how its dynamics in planta compares with that of established symplastic diffusion markers remains unknown.

A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19.

Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T-DNA is engineered with the gene(s) of interest. However, gene expression during 'agro-infiltration' is intrinsically and universally impeded by the onset of post-transcriptional gene silencing (PTGS).

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response.

HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement.

Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis.

Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference.

In RNA interference (RNAi), the RNase III Dicer processes long double-stranded RNA (dsRNA) into short interfering RNA (siRNA), which, when loaded into ARGONAUTE (AGO) family proteins, execute gene silencing. Remarkably, RNAi can act non-cell autonomously: it is graft transmissible, and plasmodesmata-associated proteins modulate its cell-to-cell spread. Nonetheless, the molecular mechanisms involved remain ill defined, probably reflecting a disparity of experimental settings.

Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues.

Arabidopsis encodes 10 ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22 nucleotide (nt) long small RNAs (sRNAs) to mediate post-transcriptional gene silencing (PTGS) or 24 nt sRNAs to promote RNA-directed DNA methylation. Using full-locus constructs, we characterized the expression, biochemical properties and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires.

Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ.

Loaded into ARGONAUTE(AGO) proteins, eukaryotic micro(mi)RNAs regulate gene expression via cleavage, translational repression, and/or accelerated decay of sequence-complementary target transcripts. Despite their importance in development, cell identity maintenance and stress responses, how individual miRNAs contribute to spatial gene regulation within the complex cell mosaics formed in tissues/organs has remained inaccessible in any organism to date. We have developed a non-invasive methodology to examine, at single-cell-type resolution, the AGO-loading and activity patterns of entire miRNA cohorts in intact organs, applied here to the Arabidopsis root tip.

Chemical enhancers of posttranscriptional gene silencing in .

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In , TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins.

Extensive profiling in reveals abundant polysome-associated 24-nt small RNAs including AGO5-dependent pseudogene-derived siRNAs.

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In , this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action.

A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding.

In , ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCF) complex and subsequent degradation.

Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants.

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations.

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export.

A genome-wide transcriptome and translatome analysis of transposons identifies a unique and conserved genome expression strategy for retroelements.

Retroelements, the prevalent class of plant transposons, have major impacts on host genome integrity and evolution. They produce multiple proteins from highly compact genomes and, similar to viruses, must have evolved original strategies to optimize gene expression, although this aspect has been seldom investigated thus far. Here, we have established a high-resolution transcriptome/translatome map for the near-entirety of transposons, using two distinct DNA methylation mutants in which transposon expression is broadly de-repressed.

A single miR390 targeting event is sufficient for triggering TAS3-tasiRNA biogenesis in Arabidopsis.

In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called 'two-hit' process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production.

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1.

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner.

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing.

Phytoviruses encode viral suppressors of RNA silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies. Comparative biochemical analyses of distinct VSRs in the reconstituted assay showed that in all cases tested, VSR interactions with siRNA duplexes inhibited the loading, but not the activity, of antiviral AGO1 and AGO2.

DNA Methylation Influences the Expression of DICER-LIKE4 Isoforms, Which Encode Proteins of Alternative Localization and Function.

Plant RNA silencing operates via RNA-directed DNA-methylation (RdDM) to repress transcription or by targeting mRNAs via posttranscriptional gene silencing (PTGS). These pathways rely on distinct Dicer-like (DCL) proteins that process double-stranded RNA (dsRNA) into small-interfering RNAs (siRNAs). Here, we explored the expression and subcellular localization of Arabidopsis thaliana DCL4.