Damien Garcia

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.

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans.

The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis

Arabidopsis seed size is regulated by the IKU pathway that includes IKU2 (a leucine-rich repeat kinase) and MINI3 (a WRKY transcription factor). We report the cloning of the IKU1 (At2g35230) gene. iku1 mutants cause reduced endosperm growth and the production of small seeds. IKU1 encodes a protein containing a VQ motif, which is a motif specific to plants. IKU1 is expressed in the early endosperm and its progenitor, the central cell. Restoration of IKU1 function in the endosperm is sufficient to rescue seed size. A genomic construct carrying mutations in the VQ motif failed to complement the iku1 mutation, suggesting an essential role for the VQ motif. IKU1 interacts with MINI3 in the yeast two-hybrid system, consistent with an IKU1 function in the IKU-MINI pathway. Our data support the proposition that endosperm development is an important determinant of seed size.

Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

Background Different classes of small RNAs (sRNAs) refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs), which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs), and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. Principal Findings We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF)-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper) side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower) side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. Conclusions/Significance Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

NERD, a Plant-Specific GW Protein, Defines an Additional RNAi-Dependent Chromatin-Based Pathway in Arabidopsis

In Arabidopsis, transcriptional gene silencing (TGS) can be triggered by 24 nt small-interfering RNAs (siRNAs) through the RNA-directed DNA methylation (RdDM) pathway. By functional analysis of NERD, a GW repeat- and PHD finger-containing protein, we demonstrate that Arabidopsis harbors a second siRNA-dependent DNA methylation pathway targeting a subset of nonconserved genomic loci. The activity of the NERD-dependent pathway differs from RdDM by the fact that it relies both on silencing-related factors previously implicated only in posttranscriptional gene silencing (PTGS), including RNA-DEPENDENT RNA POLYMERASE1/6 and ARGONAUTE2, and most likely on 21 nt siRNAs. A central role for NERD in integrating RNA silencing and chromatin signals in transcriptional silencing is supported by data showing that it binds both to histone H3 and AGO2 proteins and contributes to siRNA accumulation at a NERD-targeted locus. Our results unravel the existence of a conserved chromatin-based RNA silencing pathway encompassing both PTGS and TGS components in plants.

A miRacle in plant development: role of microRNAs in cell differentiation and patterning.

MicroRNAs (miRNAs) are endogenous small regulatory RNAs, which control gene expression in eukaryotes. In plants they repress mRNA targets containing a highly complementary site, either by cleavage or translational repression. Studies of individual miRNA/target interactions highlight the involvement of the miRNA-based regulations in a broad range of developmental programs, throughout plant lifecycle. MicroRNAs can have distinct regulatory functions on their targets: some determine their spatial accumulation, some have a buffering role that ensures the robustness of their expression pattern, and finally others establish the temporal expression of targeted genes.

Nonsense-Mediated Decay Serves as a General Viral Restriction Mechanism in Plants

Summary (+)strand RNA viruses have to overcome various points of restriction in the host to establish successful infection. In plants, this includes RNA silencing. To uncover additional bottlenecks to RNA virus infection, we genetically attenuated the impact of RNA silencing on transgenically expressed Potato virus X (PVX), a (+)strand RNA virus that replicates in Arabidopsis. A genetic screen in this sensitized background uncovered how nonsense-mediated decay (NMD), a host RNA quality control mechanism, recognizes and eliminates PVX RNAs with internal termination codons and long 3′ UTRs. NMD also operates in natural infection contexts, and while some viruses have evolved genome expression strategies to overcome this process altogether, the virulence of NMD-activating viruses entails their ability to directly suppress NMD or to promote an NMD-unfavorable cellular state. These principles of induction, evasion, and suppression define NMD as a general viral restriction mechanism in plants that also likely operates in animals.

Trans-species synthetic gene design allows resistance pyramiding and broad-spectrum engineering of virus resistance in plants

Summary To infect plants, viruses rely heavily on their hosts machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.

The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis

The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.

Accuracy of speckle tracking in the context of stress echocardiography in short axis view: An in vitro validation study

Aim This study aimed to test the accuracy of a speckle tracking algorithm to assess myocardial deformation in a large range of heart rates and strain magnitudes compared to sonomicrometry. Methods and results Using a tissue-mimicking phantom with cyclic radial deformation, radial strain derived from speckle tracking (RS-SpT) of the upper segment was assessed in short axis view by conventional echocardiography (Vivid q, GE) and post-processed with clinical software (EchoPAC, GE). RS-SpT was compared with radial strain measured simultaneously by sonomicrometers (RS-SN). Radial strain was assessed with increasing deformation rates (60 to 160 beats/min) and increasing pulsed volumes (50 to 100 ml/beat) to simulate physiological changes occurring during stress echocardiography. There was a significant correlation (R2 = 0.978, P <0.001) and a close agreement (bias ± 2SD, 0.39 ± 1.5%) between RS-SpT and RS-SN. For low strain values (<15%), speckle tracking showed a small but significant overestimation of radial strain compared to sonomicrometers. Two-way analysis of variance did not show any significant effect of the deformation rate. For RS-SpT, the feasibility was excellent and the intra- and inter-observer variability were low (the intraclass correlation coefficients were 0.96 and 0.97, respectively). Conclusions Speckle tracking demonstrated a good correlation with sonomicrometry for the assessment of radial strain independently of the heart rate and strain magnitude in a physiological range of values. Though speckle tracking seems to be a reliable and reproducible technique to assess myocardial deformation variations during stress echocardiography, further studies are mandated to analyze the impact of angulated and artefactual out-of-plane motions and inter-vendor variability.