RNA-Dependent Epigenetic Silencing Directs Transcriptional Downregulation Caused by Intronic Repeat Expansions.

Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.

Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

A Chemical Reaction Network Drives Complex Population Dynamics in Oscillating Self-Reproducing Vesicles.

We report chemically fueled oscillations of vesicles. The population cycling of vesicles is driven by their self-reproduction and collapse within a biphasic reaction network involving the interplay of molecular and supramolecular events. We studied the oscillations on the molecular and supramolecular scales and tracked vesicle populations in time by interferometric scattering microscopy and dynamic light scattering. Complex supramolecular events were observed during oscillations─including vesicle reproduction, growth, and decomposition─and differences in the number, size, and mass of aggregates can often be observed within and between pulses. This system's dynamic behavior is reminiscent of a reproductive cycle in living cells.

Superior Sagittal Sinus Thrombectomy in Pediatric Head Injury.

INTRODUCTION: Injury and subsequent thrombosis of the cerebral venous sinuses may be caused by closed head injuries secondary to a variety of different mechanisms. Skull fractures can lacerate or otherwise disrupt adjacent dural sinuses. The sequelae of such injuries may include thrombosis and either partial or total occlusion of the sinus, ultimately resulting in significant venous congestion. Sagittal sinus injury is associated with a more serious outcome due to the obligatory flow into the sinus, especially posterior to the coronal suture. In such cases, venous infarction may be a severe and life-threatening complication of head injury. CASE PRESENTATION: A 2-year-old female presented with a depressed skull fracture near the midline and a thrombus in the sagittal sinus. Anticoagulation, the standard treatment cerebral venous sinus thrombosis (CVST), was contraindicated due to intracranial hemorrhage, so immediate thrombectomy was performed with successful neurologic recovery at 9-month follow-up. To our knowledge, this case is the youngest patient documented to receive mechanical thrombectomy for superior sagittal sinus (SSS) thrombosis due to trauma. CONCLUSION: Closed head injuries in pediatric patients may be associated with CVST, with resulting venous drainage compromise and profound neurologic sequelae. Unlike adult patients with spontaneous CVST in which anticoagulation are the standard of care, pediatric patients experiencing traumatic CVST may have contraindications to anticoagulants. If the patient has a contraindication to anticoagulation such as intracranial bleeding, endovascular mechanical thrombectomy may be an effective intervention when performed by an experienced neurointerventionalist.

Catalytic Enantioselective Synthesis of 3-Piperidines from Arylboronic Acids and Pyridine.

Piperidines are frequently found in natural products and are of importance to the pharmaceutical industry. A generally useful asymmetric route to enantiomerically enriched 3-substituted piperidines remains elusive. Here we report a cross-coupling approach to enantioenriched 3-piperidines from pyridine- and sp2-hybridized boronic acids. The key step involves a Rh-catalyzed asymmetric reductive Heck reaction of aryl, heteroaryl, or vinyl boronic acids and phenyl pyridine-1(2H)-carboxylate to provide 3-substituted tetrahydropyridines in high yield and excellent enantioselectivity with a wide functional group tolerance. A three-step process involving i) partial reduction of pyridine, ii) Rh-catalyzed asymmetric carbometalation, and then iii) another reduction provides access to a wide variety of enantioenriched 3-piperidines, including clinically used materials such as Preclamol and Niraparib.

Can Craniosynostosis be Diagnosed on Physical Examination? A Retrospective Review.

Craniosynostosis is a developmental craniofacial defect in which one or more sutures of the skull fuse together prematurely. Uncorrected craniosynostosis may have serious complications including elevated intracranial pressure, developmental delay, and blindness. Proper diagnosis of craniosynostosis requires a physical examination of the head with assessment for symmetry and palpation of sutures for prominence. Often, if craniosynostosis is suspected, computed tomography (CT) imaging will be obtained. Recent literature has posited that this is unnecessary. This study aims to address whether physical examination alone is sufficient for the diagnosis and treatment planning of single suture craniosynostosis. Between 2015 and 2022, the Divisions of Pediatric Neurosurgery and Pediatric Plastic Surgery at UTHealth Houston evaluated 140 children under 36 months of age with suspected craniosynostosis by physical examination and subsequently ordered CT imaging for preoperative planning. Twenty-three patients received a clinical diagnosis of multi-sutural or syndromic craniosynostosis that was confirmed by CT. One hundred seventeen patients were diagnosed with single suture craniosynostosis on clinical examination and follow-up CT confirmed suture fusion in 109 (93.2%) patients and identified intracranial anomalies in 7 (6.0%) patients. These patients underwent surgical correction. Eight (6.8%) patients showed no evidence of craniosynostosis on CT imaging. Treatment for patients without fused sutures included molding helmets and observation alone. This evidence suggests that physical examination alone may be inadequate to accurately diagnose single suture synostosis, and surgery without preoperative CT evaluation could lead to unindicated procedures.

Rhodium-Catalyzed Asymmetric Arylation of Cyclobutenone Ketals.

Complex cyclobutanes are important motifs in both bioactive molecules and natural products, yet their enantioselective preparation has not been widely explored. In this work, we describe rhodium-catalyzed enantioselective additions of aryl and vinyl boronic acids to cyclobutenone ketals. This transformation involves enantioselective carbometalation to give cyclobutyl-rhodium intermediates, followed by ß-oxygen elimination to afford enantioenriched enol ethers. Overall, this addition serves as a surrogate for Rh-catalyzed 1,4-additions to cyclobutenone.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Back to the Beginning: An Algorithmic Approach to Neonatal Myelomeningocele Repair.

INTRODUCTION: Myelomeningoceles are formed by prenatal failure of neural tube closure and can cause hydrocephalus, motor abnormalities, and developmental delay. Although small defects are amenable to primary closure, larger defects often require complex reconstruction. Our goal was to identify factors associated with postoperative soft tissue complications and develop a systematic approach for myelomeningocele closure. METHODS: A retrospective review was performed at the Children's Memorial Hermann Hospital from January 2013 to January 2019. Patients were identified using International Classification of Diseases, Ninth Revision/Tenth Revision , codes for myelomeningocele. Cohorts were stratified by reconstruction type and defect location. Primary outcomes were incidence of complications including cerebrospinal fluid leak, superficial and deep infection, and wound dehiscence. In addition, we developed an algorithm to standardize closure approach for patients with myelomeningoceles. RESULTS: A total of 172 patients with myelomeningocele were identified with 73 patients undergoing postnatal repair. Overall, 72% of defects were >5 cm. Defects were in the lumbar (9%), sacral (8%), and junctional (83%) regions. Overall, 30.1% patients underwent lumbar myofascial repair with 39.7% requiring fasciocutaneous flaps. Larger defects (>5 cm) were more likely to be closed with complex fasciocutaneous flaps (82.8% vs 66.0%, P = 0.11). No significant differences were observed in complication rates. CONCLUSIONS: In this series, patients with larger myelomeningoceles appear to benefit from complex flap closure. We propose a 5-layer closure for patients with myelomeningocele including the routine use of a myofascial layer. Cutaneous closure technique should be tailored based on specific defect characteristics as outlined in our algorithm. This approach streamlines myelomeningocele repair while optimizing outcomes and decreasing downstream complications.

RNAi as a Foliar Spray: Efficiency and Challenges to Field Applications.

RNA interference (RNAi) is a powerful tool that is being increasingly utilized for crop protection against viruses, fungal pathogens, and insect pests. The non-transgenic approach of spray-induced gene silencing (SIGS), which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, has come to prominence due to its safety and environmental benefits in addition to its wide host range and high target specificity. However, along with promising results in recent studies, several factors limiting SIGS RNAi efficiency have been recognized in insects and plants. While sprayed dsRNA on the plant surface can produce a robust RNAi response in some chewing insects, plant uptake and systemic movement of dsRNA is required for delivery to many other target organisms. For example, pests such as sucking insects require the presence of dsRNA in vascular tissues, while many fungal pathogens are predominately located in internal plant tissues. Investigating the mechanisms by which sprayed dsRNA enters and moves through plant tissues and understanding the barriers that may hinder this process are essential for developing efficient ways to deliver dsRNA into plant systems. In this review, we assess current knowledge of the plant foliar and cellular uptake of dsRNA molecules. We will also identify major barriers to uptake, including leaf morphological features as well as environmental factors, and address methods to overcome these barriers.

Non-stabilized nucleophiles in Cu-catalysed dynamic kinetic asymmetric allylic alkylation.

The development of new reactions forming asymmetric carbon-carbon bonds has enabled chemists to synthesize a broad range of important carbon-containing molecules, including pharmaceutical agents, fragrances and polymers. Most strategies to obtain enantiomerically enriched molecules rely on either generating new stereogenic centres from prochiral substrates or resolving racemic mixtures of enantiomers. An alternative strategy--dynamic kinetic asymmetric transformation--involves the transformation of a racemic starting material into a single enantiomer product, with greater than 50 per cent maximum yield. The use of stabilized nucleophiles (pKa < 25, where Ka is the acid dissociation constant) in palladium-catalysed asymmetric allylic alkylation reactions has proved to be extremely versatile in these processes. Conversely, the use of non-stabilized nucleophiles in such reactions is difficult and remains a key challenge. Here we report a copper-catalysed dynamic kinetic asymmetric transformation using racemic substrates and alkyl nucleophiles. These nucleophiles have a pKa of ≥50, more than 25 orders of magnitude more basic than the nucleophiles that are typically used in such transformations. Organometallic reagents are generated in situ from alkenes by hydrometallation and give highly enantioenriched products under mild reaction conditions. The method is used to synthesize natural products that possess activity against tuberculosis and leprosy, and an inhibitor of para-aminobenzoate biosynthesis. Mechanistic studies indicate that the reaction proceeds through a rapidly isomerizing intermediate. We anticipate that this approach will be a valuable complement to existing asymmetric catalytic methods.

Low Transverse versus Midline Abdominal Skin Incisions for in utero Spina Bifida Repair.

OBJECTIVE: The 2 types of maternal skin incisions for in utero spina bifida repair are low transverse (LT) incision perceived to be cosmetic benefit and midline longitudinal (ML) incision, typically associated with a reduction in surgical time and lower blood loss. Our objective was to compare short- and long-term outcomes associated with these 2 types of skin incisions following in utero spina bifida repair. METHODS: Prospective observational cohort of 72 patients undergoing fetal spina bifida repair at a single institution between September 2011 and August 2018. The decision for the type of incision was at the discretion of the surgeons. The primary outcome was total operative time. Secondary outcomes included an analog scale of wound pain score on postoperative day 3, duration of postoperative stay, and postoperative wound complications within the first 4 weeks. The Patient Scar Assessment Questionnaire, a validated questionnaire, was obtained for all patients (≥6 months from delivery) using 4 categories (appearance, consciousness, satisfaction with appearance and with symptoms), with higher scores reflecting a poorer perception of the scar. RESULTS: There were 43 women (59.7%) in the LT group and 29 (40.3%) in the ML group. In all patients, the same incision was used during cesarean delivery. The total operative time was higher in the LT group by 33 min (p < 0.001), primarily due to abdominal wall incision time (open and closure). No significant differences were found between the groups in pain score, length of postoperative stay, or the rate of wound complications. Fifty-three patients (73.6%) responded to the questionnaire, 36/43 from the LT group and 17/29 from the ML group. There was no difference in the scores of appearance, consciousness, and satisfaction with appearance and symptoms between the groups. CONCLUSION: ML incisions shorten operative times without altering long-term incision-related satisfaction when compared to LT incisions.

Prenatal and Postnatal Imaging Findings After Fetal Repair of Spinal Dysraphisms Using Cryopreserved Human Umbilical Cord Patch: A Case Series of 4 Patients.

We describe a case series of imaging findings of 4 patients who underwent spinal dysraphisms repair in utero with novel patch material, cryopreserved human umbilical cord, in our institution. In our study, the prenatal and postnatal magnetic resonance imaging and ultrasound are reviewed and showed cord tethering and syrinx progression in all cases. Our report is the first description of magnetic resonance imaging and ultrasound findings in the context of using this novel patch in severe cases of spinal dysraphisms.

Outcomes and prognostic factors of pediatric patients with a Glasgow Coma Score of 3 after blunt head trauma.

PURPOSE: This study aims to assess outcomes of pediatric patients with blunt traumatic brain injury (TBI) with a presenting Glasgow Coma Score (GCS) of 3. METHODS: After local institutional review board approval, we identified patients ages 0 to15 years with blunt TBI and a reported GCS of 3 between 2007 and 2017 from a pediatric level 1 trauma center prospective registry. Exclusion criteria were cardiac death on arrival and penetrating injury. We recorded clinical variables from patients with a non-pharmacologic GCS of 3 and pupillary exam documented by a neurosurgical attending or resident. The original Glasgow Outcome Scale (GOS) was used to compare with other studies. Importance of variables to survival was calculated. RESULTS: A total of 88 patients (mean age 6.9 years) were included with a mortality rate of 68%. Twelve percent had a poor long-term outcome (GOS 2 or 3) while 20% had a good long-term outcome (GOS 4 or 5). Median follow-up was 1.8 years. Initial group comparison revealed patients in group 1 (survivors) had less hypotension on arrival (14% SBP < 90 mmHg vs. 66%, p < 0.0001), higher temperatures on arrival (36.3 °C vs 34.9 °C, p = 0.0002), lower ISS (29.7 vs 39.5, p = 0.003), less serious injury to other major organs (34% vs 61%, p = 0.02), more epidural hematomas (24% vs 7%, p = 0.04), and less evidence of brain ischemia on CT (7% vs 39%, p = 0.002) or brainstem infarct, hemorrhage, or herniation (0% vs 27%, p = 0.002). Differences between the 2 groups in age, sex, race, MOI, AIS score, presence of midline shift > 5 mm, or time from injury to hospital arrival or time to surgery were not statistically significant. Classification tree analysis showed that the most important variable for survival was pupillary exam; mortality was 92% in presence of bilateral, fixed dilated pupils. The relative importance of initial temperature, MOI, and hypotension to survivability was 0.79, 0.75, and 0.47, respectively. CONCLUSION: Twenty percent of our pediatric non-pharmacologic GCS 3 cohort had a good functional outcome. Lack of bilaterally fixed and dilated pupils was the most important factor for survival. Temperature, MOI, and hypotension also correlated with survival. The data support selective aggressive management for these patients.

Is Less Actually More? An Evaluation of Surgical Outcomes Between Endoscopic Suturectomy and Open Cranial Vault Remodeling for Craniosynostosis.

BACKGROUND: Both open cranial vault remodeling (CVR) and endoscopic suturectomy are effective in treating the anatomical deformity of craniosynostosis. While parents are increasingly knowledgeable about these 2 treatment options, information regarding the perioperative outcomes remains qualitative. This makes preoperative counseling regarding surgical choices difficult. The purpose of this study was to evaluate the outcomes in patients with craniosynostosis who underwent traditional CVR versus endoscopic suturectomy. METHODS: Open and endoscopic craniosynostosis surgeries performed at our institution from January 2014 through December 2018 were retrospectively reviewed and perioperative data, including operative time, estimated blood loss, transfusion rate and length of stay, was analyzed. A student t test was used with significance determined at P < 0.05. RESULTS: CVR was performed for 51 children while 33 underwent endoscopic procedures. Endoscopic suturectomy was performed on younger patients (3.8 versus 14.0 months, P < 0.001), had shorter operative time (70 versus 232 minutes, P < 0.001), shorter total anesthesia time (175 versus 352 minutes, P < 0.001), lower estimated blood loss (10 versus 28 ml/kg, P < 0.001), lower percentage transfused (42% versus 98%, P < 0.001), lower transfusion volume (22 versus 48 ml/kg, P < 0.001), and shorter length of stay (1.8 versus 4.1 days, P < 0.001) when compared to open CVR. CONCLUSION: Both open CVR and endoscopic suturectomy are effective in treating deformities due to craniosynostosis. The endoscopic suturectomy had significantly shorter operative and anesthesia time as well as overall and PICU length of stay. CVR was associated with greater intraoperative blood loss and more frequently required higher rates of blood transfusions.

Coupled Metabolic Cycles Allow Out-of-Equilibrium Autopoietic Vesicle Replication.

We report chemically fuelled out-of-equilibrium self-replicating vesicles based on surfactant formation. We studied the vesicles' autocatalytic formation using UPLC to determine monomer concentration and interferometric scattering microscopy at the nanoparticle level. Unlike related reports of chemically fuelled self-replicating micelles, our vesicular system was too stable to surfactant degradation to be maintained out of equilibrium. The introduction of a catalyst, which introduces a second catalytic cycle into the metabolic network, was used to close the first cycle. This shows how coupled catalytic cycles can create a metabolic network that allows the creation and perseverance of fuel-driven, out-of-equilibrium self-replicating vesicles.

SCRAM: a pipeline for fast index-free small RNA read alignment and visualization.

Summary: Small RNAs play key roles in gene regulation, defense against viral pathogens and maintenance of genome stability, though many aspects of their biogenesis and function remain to be elucidated. SCRAM (Small Complementary RNA Mapper) is a novel, simple-to-use short read aligner and visualization suite that enhances exploration of small RNA datasets. Availability and implementation: The SCRAM pipeline is implemented in Go and Python, and is freely available under MIT license. Source code, multiplatform binaries and a Docker image can be accessed via https://sfletc.github.io/scram/. Supplementary information: Supplementary data are available at Bioinformatics online.

Infusion of 5-Azacytidine (5-AZA) into the fourth ventricle or resection cavity in children with recurrent posterior Fossa Ependymoma: a pilot clinical trial.

BACKGROUND: DNA methylation inhibitors are logical therapeutic candidates for ependymomas originating in the posterior fossa of the brain. Our objective was to test the safety of infusing 5-Azacytidine (5-AZA), a DNA methylation inhibitor, directly into cerebrospinal fluid (CSF) spaces of the fourth ventricle or tumor resection cavity in children with recurrent ependymoma originating in the posterior fossa. MATERIALS AND METHODS: In patients with recurrent ependymoma whose disease originated in the posterior fossa, a maximal safe subtotal tumor resection was performed. At the conclusion of the tumor resection, a catheter was surgically placed into the fourth ventricle or tumor resection cavity and attached to a ventricular access device. CSF flow from the posterior fossa to the sacrum was confirmed by CINE phase contrast magnetic resonance imaging (MRI) postoperatively. 12 consecutive weekly 10 milligram (mg) infusions of 5-Azacytidine (AZA) were planned. Disease response was monitored with MRI scans and CSF cytology. RESULTS: Six patients were enrolled. One patient was withdrawn prior to planned 5-AZA infusions due to surgical complications after tumor resection. The remaining five patients received 8, 12, 12, 12, and 12 infusions, respectively. There were no serious adverse events or new neurological deficits attributed to 5-AZA infusions. All five patients with ependymoma who received 5-AZA infusions had progressive disease. Two of the five patients, however, were noted to have decrease in the size of at least one intraventricular lesion. CONCLUSION: 5-AZA can be infused into the fourth ventricle or posterior fossa tumor resection cavity without causing neurological toxicity. Future studies with higher doses and/or increased dosing frequency are warranted.

Controlling the Kinetics of Self-Reproducing Micelles by Catalyst Compartmentalization in a Biphasic System.

Compartmentalization of reactions is ubiquitous in biochemistry. Self-reproducing lipids are widely studied as chemical models of compartmentalized biological systems. Here, we explore the effect of catalyst location on copper-catalyzed azide-alkyne cycloadditions which drive the self-reproduction of micelles from phase-separated components. Tuning the hydrophilicity of the copper-ligand complex, so that hydro-phobic or -philic catalysts are used in combination with hydro-philic and -phobic coupling partners, provides a wide range of reactivity patterns. Analysis of the kinetic data shows that reactions with a hydrophobic catalyst are faster than with a hydrophilic catalyst. Diffusion-ordered spectroscopy experiments suggest compartmentalization of the hydrophobic catalyst inside micelles while the hydrophilic catalyst remains in the bulk aqueous phase. The autocatalytic effects observed can be tuned by varying reactant structure and coupling a hydrophilic alkyne and hydrophobic azide results in a more pronounced autocatalytic effect. We propose and test a model that rationalizes the observations in terms of the phase behavior of the reaction components and catalysts.

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.