Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a ß-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

G-U base-paired hpRNA confers potent inhibition of small RNA function in plants.

MicroRNA (miRNA) target mimicry technologies, utilizing naturally occurring miRNA decoy molecules, represent a potent tool for analyzing miRNA function. In this study, we present a highly efficient small RNA (sRNA) target mimicry design based on G-U base-paired hairpin RNA (hpG:U), which allows for the simultaneous targeting of multiple sRNAs. The hpG:U constructs consistently generate high amounts of intact, polyadenylated stem-loop (SL) RNA outside the nuclei, in contrast to traditional hairpin RNA designs with canonical base pairing (hpWT), which were predominantly processed resulting in a loop. By incorporating a 460-bp G-U base-paired double-stranded stem and a 312-576 nt loop carrying multiple miRNA target mimicry sites (GUMIC), the hpG:U construct displayed effective repression of three Arabidopsis miRNAs, namely miR165/166, miR157, and miR160, both individually and in combination. Additionally, a GUMIC construct targeting a prominent cluster of siRNAs derived from cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) effectively inhibited Y-Sat siRNA-directed silencing of the chlorophyll biosynthetic gene CHLI, thereby reducing the yellowing symptoms in infected Nicotiana plants. Therefore, the G-U base-paired hpRNA, characterized by differential processing compared to traditional hpRNA, acts as an efficient decoy for both miRNAs and siRNAs. This technology holds great potential for sRNA functional analysis and the management of sRNA-mediated diseases.

Enhanced Ionic Power Generation via Light-Driven Active Ion Transport Across 2D Semiconductor Heterostructures.

2D semiconductor heterostructures exhibit broad application prospects. However, regular nanochannels of heterostructures rarely caught the researcher's attention. Herein, a metal-organic framework (i.e., Cu3(HHTP)2) and transition metal dichalcogenides (i.e., MoS2)-based multilayer van der Waals heterostructure (i.e., Cu3(HHTP)2/MoS2) realized band alignment-dominated light-driven ion transport and further light-enhanced ionic energy generation. High-density channels of the heterostructure provide high-speed pathways for ion transmembrane transport. Upon light illumination, a net ionic flow occurs at a symmetric concentration, suggesting a directional cationic transport from Cu3(HHTP)2 to MoS2. This is because Cu3(HHTP)2/MoS2 heterostructures containing type-II band alignment can generate photovoltaic motive force through light-induced efficient charge separation to drive ion transport. After introducing into the ionic power generation system, the maximum power density under illumination can achieve notable improvement under different concentration differences. In addition to the photovoltaic motive force, type-II band alignment and material defect capture-induced surface charge increase also raise ion selectivity and flux, greatly facilitating ionic energy generation. This work demonstrates that 2D semiconductor heterostructures with rational band alignment can not only be a potential platform for optimizing light-enhanced ionic energy harvesting but also provide a new thought for biomimetic iontronic devices.

Nanofluidic Membrane-Assisted Organic Electrochemical Transistors for Bioinspired Gustatory Sensation Based on Selective Cation Transport.

Natural organisms have evolved precise sensing systems relying on unique ion channels, which can efficiently perceive various physical/chemical stimuli based on ionic signal transmission in biological fluid environments. However, it is still a huge challenge to achieve extensive applications of the artificial counterparts as an efficient wet sensing platform due to the fluidity of the working medium. Herein, nanofluidic membranes with selective cation transport properties and solid-state organic electrochemical transistors (OECTs) with amplified signals are integrated together to mimic human gustatory sensation, achieving ionic gustatory reagent recognition and a portable configuration. Cu-HHTP nanofluidic membranes with selective cation transport through their uniform micropores are constructed first, followed by assembly with OECTs to form the designed nanofluidic membrane-assisted OECTs (nanofluidic OECTs). As a result, they can distinguish typically ionic gustatory reagents, and even ionic liquids (ILs), demonstrating enhanced gustatory perception performance under a wide concentration range (10-7-10-1 m) compared with those of conventional OECTs. The linear correlations between the response and the reagent concentration further indicate the promising potential for practical application as a next-generation sensing platform. It is suggested that nanofluidic membranes mediated intramembrane cation transport based on the steric hindrance effect, resulting in distinguishable and improved response to multiple ions.

miR398b and AtC2GnT form a negative feedback loop to regulate Arabidopsis thaliana resistance against Phytophthora parasitica.

Oomycetes are diploid eukaryotic microorganisms that seriously threaten sustainable crop production. MicroRNAs (miRNAs) and corresponding natural antisense transcripts (NATs) are important regulators of multiple biological processes. However, little is known about their roles in plant immunity against oomycete pathogens. In this study, we report the identification and functional characterization of miR398b and its cis-NAT, the core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase gene (AtC2GnT), in plant immunity. Gain- and loss-of-function assays revealed that miR398b mediates Arabidopsis thaliana susceptibility to Phytophthora parasitica by targeting Cu/Zn-Superoxidase Dismutase1 (CSD1) and CSD2, leading to suppressed expression of CSD1 and CSD2 and decreased plant disease resistance. We further showed that AtC2GnT transcripts could inhibit the miR398b-CSDs module via inhibition of pri-miR398b expression, leading to elevated plant resistance to P. parasitica. Furthermore, quantitative reverse transcription PCR, RNA ligase-mediated 5'-amplification of cDNA ends (RLM-5' RACE), and transient expression assays indicated that miR398b suppresses the expression of AtC2GnT. We generated AtC2GnT-silenced A. thaliana plants by CRISPR/Cas9 or RNA interference methods, and the Nicotiana benthamiana NbC2GnT-silenced plants by virus-induced gene silencing. Pathogenicity assays showed that the C2GnT-silenced plants were more susceptible, while AtC2GnT-overexpressing plants exhibited elevated resistance to P. parasitica. AtC2GnT encodes a Golgi-localized protein, and transient expression of AtC2GnT enhanced N. benthamiana resistance to Phytophthora pathogens. Taken together, our results revealed a positive role of AtC2GnT and a negative regulatory loop formed by miR398b and AtC2GnT in regulating plant resistance to P. parasitica.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Laennec's approach for laparoscopic anatomical hemihepatectomy.

BACKGROUND: Laennec's capsule has been found for about 200 years. However, laparoscopic anatomical right and left hemihepatectomy (LARH and LALH) using Laennec's approach are rarely reported. METHODS: We retrospectively analyzed the technical details and the surgical outcomes of 15 patients who underwent LAH via Laennec's approach between May 2017 and July 2020. The operation time, intraoperative blood loss, postoperative complications, and hospital stay were recorded and analyzed. RESULTS: Four of 15 patients were diagnosed with hepatic hemangioma, 2 had hepatolithiasis, and 9 patients had primary liver cancer. During the surgery, Laennec's approach was used for LAH without conversion to open surgery. Four patients were treated with LARH, and 11 patients were cured with LALH. The mean age of the patients was 62.1 ± 6.5 years, and four were male. The mean operative time, blood loss, and length of the postoperative hospital stay were 193 ± 49 min, 247 ± 120 mL, and 8.7 ± 2.0 days, respectively. There was no incidence of postoperative bile leakage and bleeding. No mortality occurred. We also demonstrated that Laennec's capsule does exist around the peripheral hepatic veins with histological confirmation. CONCLUSIONS: Laennec's approach is safe and feasible for LAH. Precise isolation of Laennec's approach based on Laennec's capsule helps to standardize the surgical techniques for laparoscopic anatomical hepatectomy.

Comprehensive bioinformatics analysis reveals the significance of forkhead box family members in pancreatic adenocarcinoma.

BACKGROUND: Forkhead box proteins (FOXs) play important roles in multiple biological processes; while little is known regarding the role of FOX members in pancreatic adenocarcinoma (PAAD). This study aimed to comprehensively investigate the function of FOX family members in PAAD. METHODS: Expression and prognostic value of FOXs were analyzed by R language and GEPIA. Genetic alteration and promoter methylation level were analyzed using CBioPortal and UALCAN. Protein-protein interactions and gene functions were analyzed using STRING and DAVID. TIMER and SENESCopedia were utilized to analyze the correlation of FOXs with immune cell infiltration or tumor senescence. Protein levels of FOXs were detected by immunohistochemistry. RESULTS: Expression of 15 of 50 FOXs were significantly elevated in PAAD. Among these 15 differentially expressed FOXs (DE-FOXs), 4 were significantly associated with the clinical cancer stage and 4 were negatively associated with overall survival. Functions of DE-FOXs were related to epithelial tube morphogenesis, nuclear chromatin, and DNA-binding. Promoter methylation and genomic alterations were not major causes of FOX dysregulation. Most DE-FOX was correlated with diverse immune infiltration cells. Seven of the DE-FOXs were positively related to tumor senescence. The protein levels of FOXM1, FOXP1, and FOXN3 were negatively correlated with OS in the collected PAAD patients. CONCLUSIONS: FOXM1, FOXP1, and FOXN3 have prognostic value. Seven FOXs were related senescence, whereas most DE-FOXs were related to immune infiltration in PAAD. Our findings are instructive for future research on FOX family and provide novel insights into the selection of FOXs with potential prognostic or therapeutic target value.

Mutations in PpAGO3 Lead to Enhanced Virulence of Phytophthora parasitica by Activation of 25-26 nt sRNA-Associated Effector Genes.

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3, plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica, showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (QRGGYD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25-26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25-26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3 , strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25-26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica, thereby contributing to infection and pathogenicity of the pathogen.

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.

Full-Length Hairpin RNA Accumulates at High Levels in Yeast but Not in Bacteria and Plants.

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a ß-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.

DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis.

DNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:ß-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable 'quadruple' demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.

The 25-26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes.

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host-pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25-26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25-26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25-26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25-26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.