Dual FKRP/FST gene therapy normalizes ambulation, increases strength, decreases pathology, and amplifies gene expression in LGMDR9 mice.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.

Development of Assays to Measure GNE Gene Potency and Gene Replacement in Skeletal Muscle.

BACKGROUND: GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. OBJECTIVE: To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gne gene expression with human GNE in skeletal muscles. METHODS: A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNE gene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. RESULTS: Lec3 cells showed a strong deficit in MAA binding, while GNE-/-MB135 cells did not. Overexpressing GNE in Lec3 and Lec3MyoDI cells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNE stimulated human GNE expression in muscles at levels equivalent to endogenous mouse Gne at a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. CONCLUSIONS: Lec3 and Lec3MyoDI cells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNE can deliver GNE gene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.

Cation-Chloride Cotransporters KCC2 and NKCC1 as Therapeutic Targets in Neurological and Neuropsychiatric Disorders.

Neurological diseases including Alzheimer's, Huntington's disease, Parkinson's disease, Down syndrome and epilepsy, and neuropsychiatric disorders such as schizophrenia, are conditions that affect not only individuals but societies on a global scale. Current therapies offer a means for small symptomatic relief, but recently there has been increasing demand for therapeutic alternatives. The γ-aminobutyric acid (GABA)ergic signaling system has been investigated for developing new therapies as it has been noted that any dysfunction or changes to this system can contribute to disease progression. Expression of the K-Cl-2 (KCC2) and N-K-C1-1 (NKCC1) cation-chloride cotransporters (CCCs) has recently been linked to the disruption of GABAergic activity by affecting the polarity of GABAA receptor signaling. KCC2 and NKCC1 play a part in multiple neurological and neuropsychiatric disorders, making them a target of interest for potential therapies. This review explores current research suggesting the pathophysiological role and therapeutic importance of KCC2 and NKCC1 in neuropsychiatric and neurological disorders.

Therapeutic efficacy of rscAAVrh74.miniCMV.LIPA gene therapy in a mouse model of lysosomal acid lipase deficiency.

Lysosomal acid lipase deficiency (LAL-D) presents as one of two rare autosomal recessive diseases: Wolman disease (WD), a severe disorder presenting in infancy characterized by absent or very low LAL activity, and cholesteryl ester storage disease (CESD), a less severe, later onset disease form. Recent clinical studies have shown efficacy of enzyme replacement therapy for both forms of LAL-D; however, no gene therapy approach has yet been developed for clinical use. Here, we show that rscAAVrh74.miniCMV.LIPA gene therapy can significantly improve disease symptoms in the Lipa -/- mouse model of LAL-D. Treatment dramatically lowered hepatosplenomegaly, liver and spleen triglyceride and cholesterol levels, and serum expression of markers of liver damage. Measures of liver inflammation and fibrosis were also reduced. Treatment of young adult mice was more effective than treatment of neonates, and enzyme activity was elevated in serum, consistent with possible bystander effects. These results demonstrate that adeno associated virus (AAV)-mediated LIPA gene-replacement therapy may be a viable option to treat patients with LAL-D, particularly patients with CESD.

Comprehensive human amniotic fluid metagenomics supports the sterile womb hypothesis.

As metagenomic approaches for detecting infectious agents have improved, each tissue that was once thought to be sterile has been found to harbor a variety of microorganisms. Controversy still exists over the status of amniotic fluid, which is part of an immunologically privileged zone that is required to prevent maternal immune system rejection of the fetus. Due to this privilege, the exclusion of microbes has been proposed to be mandatory, leading to the sterile womb hypothesis. Since nucleic acid yields from amniotic fluid are very low, contaminating nucleic acid found in water, reagents and the laboratory environment frequently confound attempts to address this hypothesis. Here we present metagenomic criteria for microorganism detection and a metagenomic method able to be performed with small volumes of starting material, while controlling for exogenous contamination, to circumvent these and other pitfalls. We use this method to show that human mid-gestational amniotic fluid has no detectable virome or microbiome, supporting the sterile womb hypothesis.

Beta-Amyloid (Aß1-42) Increases the Expression of NKCC1 in the Mouse Hippocampus.

Alzheimer's disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation-chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl-]i by accumulating and extruding Cl-, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aß1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aß1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aß1-42, but NKCC1 expression increased 30-days post-Aß1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aß1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aß1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aß1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aß1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

Delivery of siRNA therapeutics using cowpea chlorotic mottle virus-like particles.

While highly promising in medicine, gene therapy requires delivery agents to protect and target nucleic acid therapeutics. We developed a plant viral siRNA delivery platform making use of self-assembling cowpea chlorotic mottle virus (CCMV). CCMV was loaded with siRNAs targeting GFP or FOXA1; to further enhance cell uptake and intracellular trafficking, resulting in more efficient gene knockdown, we appended CCMV with a cell penetrating peptide (CPP), specifically M-lycotoxin peptide L17E.

Delivery of mitoxantrone using a plant virus-based nanoparticle for the treatment of glioblastomas.

Mitoxatrone (MTO), an antineoplastic chemotherapeutic, has potent activity against the most common and agressive type of primary brain tumor, glioblastoma multiforme (GBM). However, its poor penetration through the blood brain barrier, and cardiotoxic side effects from systemic delivery limit its effectiveness for clinical treatment. To address these limitations, we utilize a plant virus-based nanoparticle, cowpea mosaic virus (CPMV), to deliver MTO to treat GBM. In this work, we loaded MTO into the interior cavity of CPMV (CPMV-MTO) through diffusion through its pores. We report the uptake of CPMV-MTO in glioma cells and demonstrate its cytotoxic effects in vitro as a solo therapy, and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These results reveal the potential for this plant virus-based nanoparticle platform for the treatment of GBM.

Plant viral and bacteriophage delivery of nucleic acid therapeutics.

Nucleic acid therapeutics have emerged as a powerful method for treatment of many diseases. However, the challenge lies in safe and efficient delivery of nucleic acids to their target site, as they need to cross various extracellular and intracellular barriers. Mammalian viruses have initially been favored for delivery of nucleic acid therapeutics, but safety concerns regarding their immunogenicity and potential of integration have fueled the search for alternative delivery strategies. For example, chemistry and bioengineering have led to advances in the use of nonviral vectors composed of lipids and other polymers; nevertheless, the synthetic systems often do not match the efficiency achieved using the biological systems. More recently, researchers have turned toward the development of plant viruses and bacteriophages and virus-like particles as an alternative or complementary approach. These systems unite the properties of both the viral and nonviral systems and as such are a new exciting avenue toward nucleic acid delivery. This review highlights the benefits of plant viral and bacteriophage delivery of nucleic acids and provides a summary of the current progress in research in this field. WIREs Nanomed Nanobiotechnol 2018, 10:e1487. doi: 10.1002/wnan.1487 This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

A Bioengineered Positive Control for Rapid Detection of the Ebola Virus by Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

The Ebola virus (EBOV) causes a highly virulent and deadly disease. The 2014 Ebola outbreak in West Africa was the largest in history. The rapid spread highlighted the need for a quick and accurate diagnostic method that can be employed in resource-limited conditions. In this study, we developed a probe that can be used as an internal positive control, coupled with a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to accurately detect EBOV. This RT-LAMP assay is a simple one-step reaction performed at a constant temperature, and the results can be visualized by a colorimetric change from violet to sky blue. Our assay enabled detection of 10 copies of synthetic EBOV RNA within 1 h. Compared to traditional RT-qPCR, RT-LAMP requires no sophisticated equipment, the results are easier to interpret, and they can be obtained in less time. These features make RT-LAMP an ideal method for detection of EBOV in low-resource settings.

Free-Standing, Nanopatterned Janus Membranes of Conducting Polymer-Virus Nanoparticle Arrays.

Nanostructured mesoscale materials find wide-ranging applications in medicine and energy. Top-down manufacturing schemes are limited by the smallest dimension accessible; therefore, we set out to study a bottom-up approach mimicking biological systems, which self-assemble into systems that orchestrate complex energy conversion functionalities. Inspired by nature, we turned toward protein-based nanoparticle structures formed by plant viruses, specifically the cowpea mosaic virus (CPMV). We report the formation of hierarchical CPMV nanoparticle assemblies on colloidal-patterned, conducting polymer arrays using a protocol combining colloidal lithography, electrochemical polymerization, and electrostatic adsorption. In this approach, a hexagonally close-packed array of polystyrene microspheres was assembled on a conductive electrode to function as the sacrificial colloidal template. A thin layer of conducting polypyrrole material was electrodeposited within the interstices of the colloidal microspheres and monitored in situ using electrochemical quartz crystal microbalance with dissipation (EC-QCM-D). Etching the template revealed an inverse opaline conducting polymer pattern capable of forming strong electrostatic interactions with CPMV and therefore enabling immobilization of CPMV on the surface. The CPMV-polymer films were characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Furthermore, molecular probe diffusion experiments revealed selective ion transport properties as a function of the presence of the CPMV nanoparticles on the surface. Lastly, by utilizing its electromechanical behavior, the polymer/protein membrane was electrochemically released as a free-standing film, which can potentially be used for developing high surface area cargo delivery systems, stimuli-responsive plasmonic devices, and chemical and biological sensors.

Utilizing Viral Nanoparticle/Dendron Hybrid Conjugates in Photodynamic Therapy for Dual Delivery to Macrophages and Cancer Cells.

Photodynamic therapy (PDT) is a promising avenue for greater treatment efficacy of highly resistant and aggressive melanoma. Through photosensitizer attachment to nanoparticles, specificity of delivery can be conferred to further reduce potential side effects. While the main focus of PDT is the destruction of cancer cells, additional targeting of tumor-associated macrophages also present in the tumor microenvironment could further enhance treatment by eliminating their role in processes such as invasion, metastasis, and immunosuppression. In this study, we investigated PDT of macrophages and tumor cells through delivery using the natural noninfectious nanoparticle cowpea mosaic virus (CPMV), which has been shown to have specificity for the immunosuppressive subpopulation of macrophages and also targets cancer cells. We further explored conjugation of CPMV/dendron hybrids in order to improve the drug loading capacity of the nanocarrier. Overall, we demonstrated effective elimination of both macrophage and tumor cells at low micromolar concentrations of the photosensitizer when delivered with the CPMV bioconjugate, thereby potentially improving melanoma treatment.

Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays.

The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

The exosome and trans-acting small interfering RNAs regulate cuticular wax biosynthesis during Arabidopsis inflorescence stem development.

The primary aerial surfaces of land plants are covered with a cuticle, a protective layer composed of the cutin polyester matrix and cuticular waxes. Previously, we discovered a unique mechanism of regulating cuticular wax biosynthesis during Arabidopsis (Arabidopsis thaliana) stem elongation that involves ECERIFERUM7 (CER7), a core subunit of the exosome. Because loss-of-function mutations in CER7 result in reduced expression of the wax biosynthetic gene CER3, we proposed that CER7 is involved in degrading a messenger RNA encoding a CER3 repressor. To identify this putative repressor, we performed a cer7 suppressor screen that resulted in the isolation of the posttranscriptional gene-silencing components RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, indicating that small RNAs regulate CER3 expression. To establish the identity of the effector RNA species and determine whether these RNAs control CER3 transcript levels directly, we cloned additional genes identified in our suppressor screen and performed next-generation sequencing of small RNA populations that differentially accumulate in the cer7 mutant in comparison with the wild type. Our results demonstrate that the trans-acting small interfering RNA class of small RNAs are the effector molecules involved in direct silencing of CER3 and that the expression of five additional genes (EARLY RESPONSE TO DEHYDRATION14, AUXIN RESISTANT1, a translation initiation factor SUI1 family protein, and two genes of unknown function) is controlled by both CER7 and trans-acting small interfering RNAs.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Anandamide levels fluctuate in the bovine oviduct during the oestrous cycle.

Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.

Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. RESULTS: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (rp 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). INNOVATION: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. CONCLUSION: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

Immunity and early pregnancy events: are endocannabinoids the missing link?

The success of pregnancy is dependent on a number of different cell types and signalling pathways, including immune cells which play a vital role in implantation. Immune cells express transcripts for all of the components of the endocannabinoid system, but the role of this system in the function of reproductive tract immune cells is still unclear. In this review, we present the hypothesis that the endocannabinoid signalling system is central to an endocannabinoid-immune-reproductive axis, and that it acts as the link via which immune cells exert their vital influence on implantation and foetal tolerance. Pubmed and Web of Science databases were searched for studies published since 1975 which explore the interaction between the endocannabinoid system and the immune system, the endocannabinoid system in pregnancy as well as the role of immune cells in pregnancy. There is evidence that the endocannabinoid system has established effects in several immune cell lineages including NK cells and T lymphocytes known to be crucial in the development of normal pregnancy. These effects include regulation of cytokine production, chemotaxis and proliferation. The immune system plays a critical role in placental development and foetal tolerance, achieving this through a large number of cytokines and chemokines. We conclude that there are intricate molecular interactions involved in the success of early pregnancy and that the endocannabinoid system, potentially interacting with the immune system, is a key contributor to these events.

RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis.

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).