High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing.

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)-expressing Nicotiana benthamiana 16C line, high-pressure spraying protocol, and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3' of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5' and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3'-end of the target RNA.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

Sulfur Partitioning between Glutathione and Protein Synthesis Determines Plant Growth.

Photoautotrophic organisms must efficiently allocate their resources between stress-response pathways and growth-promoting pathways to be successful in a constantly changing environment. In this study, we addressed the coordination of sulfur flux between the biosynthesis of the reactive oxygen species scavenger glutathione (GSH) and protein translation as one example of a central resource allocation switch. We crossed the Arabidopsis (Arabidopsis thaliana) GSH synthesis-depleted cadmium-sensitive cad2-1 mutant, which lacks glutamate cysteine (Cys) ligase, into the sulfite reductase sir1-1 mutant, which suffers from a significantly decreased flux of sulfur into Cys and, consequently, is retarded in growth. Surprisingly, depletion of GSH synthesis promoted the growth of the sir1-1 cad2-1 double mutant (s1c2) when compared with sir1-1 Determination of GSH levels and in vivo live-cell imaging of the reduction-oxidation-sensitive green fluorescent protein sensor demonstrated significant oxidation of the plastidic GSH redox potential in cad2-1 and s1c2 This oxidized GSH redox potential aligned with significant activation of plastid-localized sulfate reduction and a significantly higher flux of sulfur into proteins. The specific activation of the serine/threonine sensor kinase Target of Rapamycin (TOR) in cad2-1 and s1c2 was the trigger for reallocation of Cys from GSH biosynthesis into protein translation. Activation of TOR in s1c2 enhanced ribosome abundance and partially rescued the decreased meristematic activity observed in sir1-1 mutants. Therefore, we found that the coordination of sulfur flux between GSH biosynthesis and protein translation determines growth via the regulation of TOR.

Functional and topological characteristics of mammalian regulatory domains.

Long-range regulatory interactions play an important role in shaping gene-expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organization where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles.

TRACER: a resource to study the regulatory architecture of the mouse genome.

BACKGROUND: Mammalian genes are regulated through the action of multiple regulatory elements, often distributed across large regions. The mechanisms that control the integration of these diverse inputs into specific gene expression patterns are still poorly understood. New approaches enabling the dissection of these mechanisms in vivo are needed. RESULTS: Here, we describe TRACER (http://tracerdatabase.embl.de), a resource that centralizes information from a large on-going functional exploration of the mouse genome with different transposon-associated regulatory sensors. Hundreds of insertions have been mapped to specific genomic positions, and their corresponding regulatory potential has been documented by analysis of the expression of the reporter sensor gene in mouse embryos. The data can be easily accessed and provides information on the regulatory activities present in a large number of genomic regions, notably in gene-poor intervals that have been associated with human diseases. CONCLUSIONS: TRACER data enables comparisons with the expression pattern of neighbouring genes, activity of surrounding regulatory elements or with other genomic features, revealing the underlying regulatory architecture of these loci. TRACER mouse lines can also be requested for in vivo transposition and chromosomal engineering, to analyse further regions of interest.

Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

Small RNA-seq dataset of wild type and 16C Nicotiana benthamiana leaves sprayed with naked dsRNA using the high-pressure spraying technique.

Double-stranded RNA (dsRNA) applications have emerged as promising alternatives to chemical plant pesticides. It has been proposed that the protective effect of dsRNA is mediated by the RNA interference (RNAi) mechanism. Small RNAs (sRNAs) are one of the landmarks of RNAi mechanisms. Two classes of sRNAs appear upon RNAi, triggered by dsRNA: The cleavage products of the dsRNA mapping directly to the dsRNA sequence and the transitive sRNAs mapping to the target transcript outside of the dsRNA sequence. Therefore, the sRNA-seq data obtained from dsRNA-treated plants have been exclusively analysed in the context of the target genes and the outcome has been considered essential to evaluate the underlying mechanism of dsRNA mediated plant protection. Using high-pressure spraying technology (HPST), we have applied a GFP targeting 139bp-long dsRNA on wild type (WT) and GFP expressing (16C) Nicotiana benthamiana plants in biological triplicates. As a control, we applied water with HPST on 16C N. benthamiana. We have acquired sRNA-seq data on the treated and control leaves 5 days post spraying. In this dataset, we have expanded our sRNA-seq analysis from the target GFP transgene sequence to the whole transcriptome of N. benthamiana to provide the community with a resource for the small RNA landscape after high-pressure spraying in 16C and WT samples. Furthermore, we have provided a comparison of sRNA landscape between WT and 16C lines.

High-Pressure-Sprayed Double Stranded RNA Does Not Induce RNA Interference of a Reporter Gene.

In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs). SiRNAs are loaded onto ARGONAUTE proteins to constitute the RNA-induced silencing complex (RISC). Natural dsRNAs derive from transcription of inverted repeats or of specific RNA molecules that are transcribed by RNA-directed RNA polymerase 6 (RDR6). Moreover, replication of infecting viruses/viroids results in the production of dsRNA intermediates that can serve as substrates for DCLs. The high effectiveness of RNAi both locally and systemically implicated that plants could become resistant to pathogens, including viruses, through artificial activation of RNAi by topical exogenous application of dsRNA. The most preferable procedure to exploit RNAi would be to simply spray naked dsRNAs onto mature plants that are specific for the attacking pathogens serving as a substitute for pesticides applications. However, the plant cell wall is a difficult barrier to overcome and only few reports claim that topical application of naked dsRNA triggers RNAi in plants. Using a transgenic Nicotiana benthamiana line, we found that high-pressure-sprayed naked dsRNA did not induce silencing of a green fluorescence protein (GFP) reporter gene. Small RNA sequencing (sRNA-seq) of the samples from dsRNA sprayed leaves revealed that the dsRNA was, if at all, not efficiently processed into siRNAs indicating that the dsRNA was insufficiently taken up by plant cells.

The Recovery from Sulfur Starvation Is Independent from the mRNA Degradation Initiation Enzyme PARN in Arabidopsis.

When plants are exposed to sulfur limitation, they upregulate the sulfate assimilation pathway at the expense of growth-promoting measures. Upon cessation of the stress, however, protective measures are deactivated, and growth is restored. In accordance with these findings, transcripts of sulfur-deficiency marker genes are rapidly degraded when starved plants are resupplied with sulfur. Yet it remains unclear which enzymes are responsible for the degradation of transcripts during the recovery from starvation. In eukaryotes, mRNA decay is often initiated by the cleavage of poly(A) tails via deadenylases. As mutations in the poly(A) ribonuclease PARN have been linked to altered abiotic stress responses in Arabidopsis thaliana, we investigated the role of PARN in the recovery from sulfur starvation. Despite the presence of putative PARN-recruiting AU-rich elements in sulfur-responsive transcripts, sulfur-depleted PARN hypomorphic mutants were able to reset their transcriptome to pre-starvation conditions just as readily as wildtype plants. Currently, the subcellular localization of PARN is disputed, with studies reporting both nuclear and cytosolic localization. We detected PARN in cytoplasmic speckles and reconciled the diverging views in literature by identifying two PARN splice variants whose predicted localization is in agreement with those observations.

Long-range enhancers regulating Myc expression are required for normal facial morphogenesis.

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital malformations observed in humans, with 1 occurrence in every 500-1,000 births. A 640-kb noncoding interval at 8q24 has been associated with increased risk of non-syndromic CL/P in humans, but the genes and pathways involved in this genetic susceptibility have remained elusive. Using a large series of rearrangements engineered over the syntenic mouse region, we show that this interval contains very remote cis-acting enhancers that control Myc expression in the developing face. Deletion of this interval leads to mild alteration of facial morphology in mice and, sporadically, to CL/P. At the molecular level, we identify misexpression of several downstream genes, highlighting combined impact on the craniofacial developmental network and the general metabolic capacity of cells contributing to the future upper lip. This dual molecular etiology may account for the prominent influence of variants in the 8q24 region on human facial dysmorphologies.

Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor.

We present here a Sleeping Beauty-based transposition system that offers a simple and efficient way to investigate the regulatory architecture of mammalian chromosomes in vivo. With this system, we generated several hundred mice and embryos, each with a regulatory sensor inserted at a random genomic position. This large sampling of the genome revealed the widespread presence of long-range regulatory activities along chromosomes, forming overlapping blocks with distinct tissue-specific expression potentials. The presence of tissue-restricted regulatory activities around genes with widespread expression patterns challenges the gene-centric view of genome regulation and suggests that most genes are modulated in a tissue-specific manner. The local hopping property of Sleeping Beauty provides a dynamic approach to map these regulatory domains at high resolution and, combined with Cre-mediated recombination, allows for the determination of their functions by engineering mice with specific chromosomal rearrangements.