David Windels

miR393 and Secondary siRNAs Regulate Expression of the TIR1/AFB2 Auxin Receptor Clade and Auxin-Related Development of Arabidopsis Leaves

The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.

The biosynthetic pathways and biological scopes of plant small RNAs.

RNA silencing plays a crucial role in coordinating the expression, stability, protection and inheritance of eukaryotic genomes. It comprises several mechanisms that invariably depend on core small RNA molecules (sRNAs) and that achieve dedicated sequence-specific functions. Biogenesis and function of sRNAs generally follow a framework that involves main protein families conserved among eukaryotes. The most recent studies have provided structural insights into the function of RNA silencing components, identified novel players of these pathways and highlighted possible emerging classes of sRNA regulators. In this review, we integrate these studies to provide updated views on the biosynthetic pathways and dedicated functions of the endogenous sRNA classes of plants, emphasizing their specialized scopes.

Role of microRNAs and other sRNAs of plants in their changing environments

Plants constantly face a complex array of environmental biotic and abiotic stimuli. Recent studies in various plants have highlighted the key roles of microRNAs and of different siRNA classes in the post-transcriptional regulation of plant genes essential for conserved responses of plants to individual stress conditions. It is not yet clear how these different signals and responses are integrated in nature. In the present review, we summarize current knowledge on sRNA-mediated responses to stress, and highlight possible directions of future research.

Primary and secondary siRNAs in geminivirus-induced gene silencing.

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

Specific impact of tobamovirus infection on the Arabidopsis small RNA profile

Tobamoviruses encode a silencing suppressor that binds small RNA (sRNA) duplexes in vitro and supposedly in vivo to counteract antiviral silencing. Here, we used sRNA deep-sequencing combined with transcriptome profiling to determine the global impact of tobamovirus infection on Arabidopsis sRNAs and their mRNA targets. We found that infection of Arabidopsis plants with Oilseed rape mosaic tobamovirus causes a global size-specific enrichment of miRNAs, ta-siRNAs, and other phased siRNAs. The observed patterns of sRNA enrichment suggest that in addition to a role of the viral silencing suppressor, the stabilization of sRNAs might also occur through association with unknown host effector complexes induced upon infection. Indeed, sRNA enrichment concerns primarily 21-nucleotide RNAs with a 5′-terminal guanine. Interestingly, ORMV infection also leads to accumulation of novel miRNA-like sRNAs from miRNA precursors. Thus, in addition to canonical miRNAs and miRNA*s, miRNA precursors can encode additional sRNAs that may be functional under specific conditions like pathogen infection. Virus-induced sRNA enrichment does not correlate with defects in miRNA-dependent ta-siRNA biogenesis nor with global changes in the levels of mRNA and ta-siRNA targets suggesting that the enriched sRNAs may not be able to significantly contribute to the normal activity of pre-loaded RISC complexes. We conclude that tobamovirus infection induces the stabilization of a specific sRNA pool by yet unknown effector complexes. These complexes may sequester viral and host sRNAs to engage them in yet unknown mechanisms involved in plant:virus interactions.

Introns of plant pri‐miRNAs enhance miRNA biogenesis

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop‐off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5′ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen‐triggered accumulation of miR163 and proper regulation of at least one of its targets.

miR393: integrator of environmental cues in auxin signaling?

Auxin signaling and plant development depend on a family of partially redundant F-box receptors of the TIR1/AFB2 Auxin Receptor (TAAR) clade. We have recently shown that the post-transcriptional regulation of the Arabidopsis thaliana TAAR gene family invokes complex sRNA regulations during development. In leaves, the microRNA miR393 appears 1) to be primarily generated from one of the two genes, AtMIR393B, 2) to regulate the expression of all four members of the clade and 3) to initiate the formation of functional secondary siRNAs, named siTAARs, from TAAR transcripts themselves. Strikingly, mir393b-1 mutants, which are impaired in the biogenesis of miR393b and siTAARs, exhibit rather mild developmental defects. The known roles of miR393 in nitrate response, in defense against pathogenic bacteria and in plant development lead us to hypothesize that miR393 plays an important role to integrate complex environmental stimuli.

miR393 is required for production of proper auxin signalling outputs.

Auxins are crucial for plant growth and development. Auxin signalling primarily depends on four partially redundant F-box proteins of the TIR1/AFB2 Auxin Receptor (TAAR) clade to trigger the degradation of AUX/IAA transcriptional repressors. Auxin signalling is a balanced system which involves complex feedback regulations. miR393 regulation of TAAR genes is important for different developmental programs and for responses to environment. However, so far, the relevance of the two MIR393 genes for Arabidopsis leaf development and their significance for auxin signalling homeostasis have not been evaluated. First, our analyses of mir393a-1 and mir393b-1 mutants and of mir393ab double mutant show that the two genes have only partially redundant functions for leaf development. Expression analyses of typical auxin-induced reporter genes have shown that the loss of miR393 lead to several unanticipated changes in auxin signalling. The expression of DR5pro:GUS is decreased, the expression of primary AUX/IAA auxin-responsive genes is slightly increased and the degradation of the AXR3-NT:GUS reporter protein is delayed in mir393ab mutants. Additional analyses using synthetic auxin and auxin antagonists indicated that miR393 deficient mutants have higher levels of endogenous AUX/IAA proteins, which in turn create a competition for degradation. We propose that the counter-intuitive changes in the expression of AUX/IAA genes and in the accumulation of AUX/IAA proteins are explained by the intrinsic nature of AUX/IAA genes which are feedback regulated by the AUX/IAA proteins which they produce. Altogether our experiments provide an additional highlight of the complexity of auxin signaling homeostasis and show that miR393 is an important component of this homeostasis.

HISTONE DEACETYLASE6 Controls Gene Expression Patterning and DNA Methylation-Independent Euchromatic Silencing

Mutation in a highly conserved domain of a histone deacetylase releases euchromatic silencing without affecting DNA methylation. To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides.

cDNA-AFLP display for the isolation of differentially expressed genes during chicory root development.

To identify genes expressed during root enlargment during the early vegetative growth of chicory (Cichorium intybus L.), we used the cDNA-AFLP technology. The radial pattern of chicory roots was investigated by histological analysis to determine the spatial vascular cambium setting. In young plantlets, serial root sections showed that differentiation of secondary tissues occurred along a gradient extending from the apex to the crown. The cDNA-AFLP technique was carried out on total mRNAs extracted from root tissues producing secondary structures and root tissues developing primary structures only. This study reports on the isolation of two transcript-derived fragments (TDFs) referred to as Y-16 and Y-21. Sequence analysis at the protein level showed that Y-16 carries a sequence highly homologous (93% identities) to the amino acid transporter-like protein 1 (AATL 1) from Arabidopsis and Y-21 presents 72% identity to AAD25141.1 Arabidopsis protein. The expression pattern of both these TDFs was analysed by northern blot and showed an over-expression during early development of chicory roots.