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.     

The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato

The plant protein ARGONAUTE1 (AGO1) functions in multiple RNA-silencing pathways, including those of microRNAs, key regulators of growth and development. Genetic analysis of ago1 mutants with informative defects has provided valuable insights into AGO1’s biological functions. Tomato encodes two AGO1 homologs (SlAGO1s), but mutants have not been described to date. To analyze SlAGO1s’ involvement in development, we confirmed that both undergo decay in the presence of the Polerovirus silencing suppressor P0 and produce a transgenic responder line (OP:P0HA) that, upon transactivation, expresses P0 C-terminally fused to a hemagglutinin (HA) tag (P0HA) and destabilizes SlAGO1s at the site of expression. By crossing OP:P0HA with a battery of driver lines, constitutive as well as organ- and stage-specific SlAGO1 downregulation was induced in the F1 progeny. Activated plants exhibited various developmental phenotypes that partially overlapped with those of Arabidopsis ago1 mutants. Plants that constitutively expressed P0HA had reduced SlAGO1 levels and increased accumulation of miRNA targets, indicating compromised SlAGO1-mediated silencing. Consistent with this, they exhibited pleiotropic morphological defects and their growth was arrested post-germination. Transactivation of P0HA in young leaf and floral organ primordia dramatically modified corresponding organ morphology, including the radialization of leaflets, petals and anthers, suggesting that SlAGO1s’ activities are required for normal lateral organ development and polarity. Overall, our results suggest that the OP:P0HA responder line can serve as a valuable tool to suppress SlAGO1 silencing pathways in tomato. The suppression of additional SlAGOs by P0HA and its contribution to the observed phenotypes awaits investigation.     

Polerovirus protein P0 prevents the assembly of small RNA‐containing RISC complexes and leads to degradation of ARGONAUTE1

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double‐stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA‐induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre‐programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high‐molecular‐weight complex, suggesting the existence of a multi‐protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co‐expressed AGO1 in both the presence and absence of P0.     

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.     

The role of ARGONAUTE1 (AGO1) in meristem formation and identity

The ARGONAUTE gene family is involved in the regulation of gene expression via the RNAi Silencing Complex (RISC). microRNA (miRNA) are 20-22bp RNAs that direct RISC to target genes. Several miRNA have been characterized in plants. Their roles include control of flowering time, floral organ identity, cell division patterns, and leaf polarity. ARGONAUTE1 (AGO1) is required for stem cell function and organ polarity, as is the closely related protein PINHEAD/ZWILLE (PNH/ZLL). Through phenotypic and double mutant analysis, we show that AGO1 regulates stem cell function via SHOOT MERISTEMLESS (STM). CUPSHAPED COTYLEDONS1 and 2 (CUC1 and CUC2) positively regulate STM and are targets of miRNA. The effect of AGO1 on leaf polarity is dependent, in part, on its role in meristem function revealed by interactions with ASYMMETRIC LEAVES1(AS1). AGO1 is required for full expression of LEAFY (LFY), APETALA1 (AP1) and AGAMOUS (AG). Flowering time is unaffected but floral meristem identity is partially restored in a curlyleaf (clf) background and this is not due to clf's affects on AG expression. CLF is over expressed in ago1, showing that the RNAi pathway regulates polycomb-type epigenetic modifiers.     

The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing

In plants RNA silencing is a host defense mechanism against viral infection, in which double‐strand RNA is processed into 21–24‐nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence‐specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single‐strand and double‐strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21‐ and 24‐nt siRNA accumulation, respectively. Unexpectedly, cell‐to‐cell movement and systemic propagation of silencing were enhanced in P1‐expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4‐dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4‐dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter‐defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity.