Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis

Formation of trans-acting small interfering RNAs (ta-siRNAs) from the TAS3 precursor is triggered by the AGO7/miR390 complex, which primes TAS3 for conversion into double-stranded RNA by the RNA-dependent RNA polymerase RDR6 and SGS3. These ta-siRNAs control several aspects of plant development. The mechanism routing AGO7-cleaved TAS3 precursor to RDR6/SGS3 and its subcellular organization are unknown. We show that AGO7 accumulates together with SGS3 and RDR6 in cytoplasmic siRNA bodies that are distinct from P-bodies. siRNA bodies colocalize with a membrane-associated viral protein and become positive for stress-granule markers upon stress-induced translational repression, this suggests that siRNA bodies are membrane-associated sites of accumulation of mRNA stalled during translation. AGO7 congregates with miR390 and SGS3 in membranes and its targeting to the nucleus prevents its accumulation in siRNA bodies and ta-siRNA formation. AGO7 is therefore required in the cytoplasm and membranous siRNA bodies for TAS3 processing, revealing a hitherto unknown role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO action in plants.     

Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway

Plants leaves develop proximodistal, dorsoventral (adaxial-abaxial), and mediolateral patterns following initiation. The Myb domain gene PHANTASTICA (PHAN) is required for adaxial fate in many plants , but the Arabidopsis ortholog ASYMMETRIC LEAVES1 (AS1) has milder effects, suggesting that alternate or redundant pathways exist . We describe enhancers of as1 with more elongate and dissected leaves. As well as RDR6, an RNA-dependent RNA polymerase previously proposed to influence as1 through microRNA , these enhancers disrupt ARGONAUTE7 (AGO7)/ZIPPY, SUPPRESSOR OF GENE SILENCING3 (SGS3), and DICER-LIKE4 (DCL4), which instead regulate trans-acting small interfering RNA (ta-siRNA) . Microarray analysis revealed that the AUXIN RESPONSE FACTOR genes ETTIN (ETT)/ARF3 and ARF4 were upregulated in ago7, whereas FILAMENTOUS FLOWER (FIL) was upregulated only in as1 ago7 double mutants. RDR6 and SGS3 likewise repress these genes, which specify abaxial fate . We show that the trans-acting siRNA gene TAS3, which targets ETT and ARF4, is expressed in the adaxial domain, and ett as1 ago7 triple mutants resemble as1. Thus FIL is downregulated redundantly by AS1 and by TAS3, acting through ETT, revealing a role for ta-siRNA in leaf polarity. RDR6 and DCL4 are required for systemic silencing, perhaps implicating ta-siRNA as a mobile signal.     

p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.     

ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha

In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking. To investigate the role of these molecules in axonogenesis, we expressed ARNO and ARF6 in cultured rat hippocampal neurons. Expression of catalytically inactive ARNO or dominant negative ARF6 resulted in enhanced axonal extension and branching and this effect was abrogated by coexpression of constitutively active ARF6. We sought to identify the downstream effectors of ARF6 during neurite extension by coexpressing phosphatidyl-inositol-4-phosphate 5-Kinase alpha [PI(4)P 5-Kinase alpha] with catalytically inactive ARNO and dominant negative ARF6. We found that PI(4)P 5-Kinase alpha plays a role in neurite extension and branching downstream of ARF6. Also, expression of inactive ARNO/ARF6 depleted the actin binding protein mammalian ena (Mena) from the growth cone leading edge, indicating that these effects on axonogenesis may be mediated by changes in cytoskeletal dynamics. These results suggest that ARNO and ARF6, through PI(4)P 5-Kinase alpha, regulate axonal elongation and branching during neuronal development.     

miR390, Arabidopsis TAS3 tasiRNAs,and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating Lateral Root Growth

Plants adapt to different environmental conditions by constantly forming new organs in response to morphogenetic signals. Lateral roots branch from the main root in response to local auxin maxima. How a local auxin maximum translates into a robust pattern of gene activation ensuring the proper growth of the newly formed lateral root is largely unknown. Here, we demonstrate that miR390, TAS3-derived trans-acting short-interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORS (ARFs) form an auxin-responsive regulatory network controlling lateral root growth. Spatial expression analysis using reporter gene fusions, tasi/miRNA sensors, and mutant analysis showed that miR390 is specifically expressed at the sites of lateral root initiation where it triggers the biogenesis of tasiRNAs. These tasiRNAs inhibit ARF2, ARF3, and ARF4, thus releasing repression of lateral root growth. In addition, ARF2, ARF3, and ARF4 affect auxin-induced miR390 accumulation. Positive and negative feedback regulation of miR390 by ARF2, ARF3, and ARF4 thus ensures the proper definition of the miR390 expression pattern. This regulatory network maintains ARF expression in a concentration range optimal for specifying the timing of lateral root growth, a function similar to its activity during leaf development. These results also show how small regulatory RNAs integrate with auxin signaling to quantitatively regulate organ growth during development.     

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.     

TRANS-ACTING SIRNA3-derived short interfering RNAs confer cleavage of mRNAs in rice

Plant TRANS-ACTING SIRNA3 (TAS3)-derived short interfering RNAs (siRNAs) include tasiR-AUXIN RESPONSE FACTORs (ARFs), which are functionally conserved in targeting ARF genes, and a set of non-tasiR-ARF siRNAs, which have rarely been studied. In this study, TAS3 siRNAs were systematically characterized in rice (Oryza sativa). Small RNA sequencing results showed that an overwhelming majority of TAS3 siRNAs belong to the non-tasiR-ARF group, while tasiR-ARFs occupy a diminutive fraction. Phylogenetic analysis of TAS3 genes across dicot and monocot plants revealed that the siRNA-generating regions were highly conserved in grass species, especially in the Oryzoideae. Target genes were identified for not only tasiR-ARFs but also non-tasiR-ARF siRNAs by analyzing rice Parallel Analysis of RNA Ends datasets, and some of these siRNA–target interactions were experimentally confirmed using tas3 mutants generated by genome editing. Consistent with the de-repression of target genes, phenotypic alterations were observed for mutants in three TAS3 loci in comparison to wild-type rice. The regulatory role of ribosomes in the TAS3 siRNA–target interactions was further revealed by the fact that TAS3 siRNA-mediated target cleavage, in particular tasiR-ARFs targeting ARF2/3/14/15, occurred extensively in rice polysome samples. Altogether, our study sheds light into TAS3 genes in plants and expands our knowledge about rice TAS3 siRNA–target interactions.

A population of rice short interfering RNAs derived from TRANS-ACTING SIRNA3 loci direct target mRNA cleavage, revealing a broader role of TRANS-ACTING SIRNA3 genes in rice growth and stress responses.     

Arabidopsis ENOR3 regulates RNAi-mediated antiviral defense

Viruses can infect host plants to cause severe diseases and substantial agricultural loss, while plants have evolved RNA interference (RNAi) strategy to defend against viral infection. Despite enormous efforts, only a few host proteins in RNAi pathway were shown to mediate antiviral defense, including RNA-dependent RNA polymerase 1 (RDR1), RDR6, DICER-LIKE 2 (DCL2) and DCL4. In this study, we carried out a genetic screen for antiviral factors of RNAi pathway in Arabidopsis rdr6 background via inoculation with a 2b-deficient Cucumber Mosaic Virus (CMV-Δ2b). We identified a mutant susceptible to CMV-Δ2b, referred to as enhancer of rdr6 (enor) 3-1 rdr6, and found that ENOR3 encodes a functionally unknown protein with high homology to the mammalian Non Imprinted in Prader-Willi/Angelman (NIPA) magnesium transporters. ENOR3 inhibits accumulation of CMV-Δ2b and acts additively with RDR1, RDR6, DCL2 and DCL4 in antiviral defense. These results uncover that ENOR3 is a key component in antiviral RNAi pathway, and provide new insights into antiviral immunity.