RNA Interference-mediated Silencing Research Articles

Gut bacterium Burkholderia cepacia (BsNLG8) and immune gene Defensin A contribute to the resistance against Nicotine-induced stress in Nilaparvata lugens (Stål)

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts.

RNA interference-mediated silencing of coat protein II (COPII) genes affects the gut homeostasis and cuticle development in Locusta migratoria

The coat protein II (COPII) complex consists of five primary soluble proteins, namely the small GTP-binding protein Sar1, the inner coat Sec23/Sec24 heterodimers, and the outer coat Sec13/Sec31 heterotetramers. COPII is essential for cellular protein and lipid trafficking through cargo sorting and vesicle formation at the endoplasmic reticulum. However, the roles of COPII assembly genes remain unknown in insects. In present study, we identified five COPII assembly genes (LmSar1, LmSec23, LmSec24, LmSec13 and LmSec31) in Locusta migratoria. RT-qPCR results revealed that these genes showed different expression patterns in multiple tissues and developmental days of fifth-instar nymphs. Injection of double-stranded RNA against each LmCOPII gene induced a high RNAi efficiency, and considerably suppressed feeding, and increased mortality to 100 %. Results from the micro-sectioning and hematoxylin–eosin staining of midguts showed that the brush border was severely damaged and the number of columnar cells was significantly reduced in dsLmCOPII-injected nymphs, as compared with the control. The dilated endoplasmic reticulum phenotype of columnar cells was observed by transmission electron microscopy. RT-qPCR results further indicated that silencing any of the five genes responsible for COPII complex assembly repressed the expression of genes involved in insulin/mTOR-associated nutritional pathway. Therefore, COPII assembly genes could be promising RNAi targets for insect pest management by disrupting gut and cuticle development.

Elimination of virus-like particles reduces protein aggregation and extends replicative lifespan in Saccharomyces cerevisiae

A major consequence of aging and stress, in yeast to humans, is an increased accumulation of protein aggregates at distinct sites within the cells. Using genetic screens, immunoelectron microscopy, and three-dimensional modeling in our efforts to elucidate the importance of aggregate annexation, we found that most aggregates in yeast accumulate near the surface of mitochondria. Further, we show that virus-like particles (VLPs), which are part of the retrotransposition cycle of Ty elements, are markedly enriched in these sites of protein aggregation. RNA interference-mediated silencing of Ty expression perturbed aggregate sequestration to mitochondria, reduced overall protein aggregation, mitigated toxicity of a Huntington's disease model, and expanded the replicative lifespan of yeast in a partially Hsp104-dependent manner. The results are in line with recent data demonstrating that VLPs might act as aging factors in mammals, including humans, and extend these findings by linking VLPs to a toxic accumulation of protein aggregates and raising the possibility that they might negatively influence neurological disease progression.

Fatty Acid Elongase Gene PcELO7 is Essential for Lipid Accumulation and Fecundity of Panonychus citri (Acari: Tetranychidae).

RNA interference (RNAi) has been proposed as a promising strategy for sustainable and ecofriendly pest control. The insect cuticle lipids were deposited on the body surface and functioned as a defense against chemical xenobiotics. They consisted of aliphatic compounds, including free fatty acids (FFAs). However, elongase of very long chain fatty acids (ELOs) is essential for FFA biosynthesis; the function of ELO is still unknown in many arthropods, including Panonychus citri (P. citri). In this study, three ELOs were cloned. Developmental-specific mRNA expression results revealed that three PcELOs were highly expressed in egg and adult females. Whereas PcELO7 was dominantly expressed in adult females. Under spirobudiclofen stress, ELOs mRNA expression had different changes, and PcELO7 was down-regulated. The silencing of PcELO7 resulted in a dramatic reduction of oviposition and hatchability. Significant reduction of FFA contents was also examined within PcELO7-repressed P. citri. In addition, we found that PcELO7 mRNA levels were related to fecundity and could affect triacylglycerol (TG) contents. The findings demonstrated that the introduction of dsPcELO7 via oral feeding induced the RNA interference-mediated silencing of a special target gene and could result in mortality and reproduction. In conclusion, PcELO7 is a special RNAi target for P. citri control, and its lethal mechanism might be disturbing lipids biosynthesis.

RAC1b Collaborates with TAp73α-SMAD4 Signaling to Induce Biglycan Expression and Inhibit Basal and TGF-β-Driven Cell Motility in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-β. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-β pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-β1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-β signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-β1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-β signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway.

Challenges and Opportunities in Plant Disease Management: A Brief Review

Plant diseases can reduce human food availability. Modern plant disease management faces problems due to climate change, fungicide resistance, pesticide residues and biodiversity loss. This review discusses problems and challenges in plant disease management and future research needs for effective management. Plant disease forecasting models can be used to predict plant diseases ahead of time. Protected cultivation combats climate change. It is necessary to find more evidence that plant pathogen diversity has a dilution effect on disease incidence. Deep learning-based disease diagnosis will help detect diseases faster. More hybrid fungicides should be developed to minimize fungicidal resistance problems. Among the molecular methods of plant disease management, RNA interference-mediated gene silencing and genome editing with CRISPR is more promising in plant disease management.

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

LC‒MS/MS and transcriptome analyses reveal saliva components of the seed-feeding truebug Pyrrhocoris apterus

Saliva secretion is essential for successful feeding by piercing–sucking insects. Despite extensive studies of the saliva composition of phloem- and blood-feeding insects, less is known about the oral secretions of seed-feeding insects. The firebug Pyrrhocoris apterus is a polyphagous insect that feeds primarily on dry seeds of various plant species. Here, liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) was used to identify 165 P. apterus salivary proteins, including 110 gelling-specific proteins, 24 watery-specific proteins, and 31 proteins common to both groups. Transcriptomic analysis of saliva glands identified 567 potential salivary proteins, of which 87 overlapped with those detected by LC‒MS/MS. Comparative analysis showed that 98 out of 165 (59.4%) P. apterus saliva proteins were associated with extra-oral digestion and detoxification and had homologs in ten other species of arthropods examined. In addition, 67 proteins were specific to P. apterus, of which 22 proteins were defined with unknown functions. RNA interference-mediated gene silencing assays indicated that P. apterus-specific proteins were vital for P. apterus survival. Our findings shed light on the function of salivary proteins in feeding by seed-feeding insects and the evolution of feeding habits in piercing–sucking insects.

Unveiling Nilaparvata lugens Stål Genes Defining Compatible and Incompatible Interactions with Rice through Transcriptome Analysis and Gene Silencing.

The brown planthopper (Nilaparvata lugens Stål, BPH) is a major pest of rice (Oryza sativa L.), causing severe crop loss. Multiple biotypes and emerging populations of BPH pose a bigger challenge for the infestations control. Although several studies have been conducted to understand the molecular mechanisms of rice-BPH interactions, there are few studies dedicated to the Indian sub-continent BPH biotype (biotype 4). Here, we analyzed the transcriptomic, physiological, and gene-silencing responses of the BPH biotype 4 during the compatible (fed on susceptible Taichung Native 1, TN1 rice) and incompatible (fed on resistant PTB33 rice) rice-BPH interactions. In the incompatible interaction, a significant reduction in the honeydew production and negative weight gain were observed in the BPH. Similarly, the trehalose and glucose contents were found to be significantly high and low, respectively, during the incompatible rice-BPH interaction. The comparative BPH transcriptome analysis identified 1875 differentially expressive genes (DEGs) between the compatible and incompatible interactions from which many were annotated to be involved in vital BPH physiological processes, including cuticle development, sugar metabolism, detoxification, molting, and xenobiotics metabolism. The RNA interference-mediated independent silencing of three selected genes, including NlCP1, NlCYP320a1, and NlTret1, revealed that these genes are important for BPH physiology and survival. Moreover, the results of this study provide valuable insights into the rice-BPH interactions involving the BPH biotype 4.

Liver factor B silencing to cure C3 glomerulopathy: Evidence from a mouse model of complement dysregulation

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease.Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely.Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.

Tweedle gene family of Drosophila suzukii (Matsumura) larva enhances the basal tolerance to cold and hypoxia.

Drosophila suzukii (Matsumura) is considered a quarantine pest in the A2 list because it causes serious infection and huge economic losses. Cold and controlled atmosphere treatments have been used to control immature stage pests in fresh fruits. Herein, the basal tolerance response of D. suzukii egg, larva and pupa to cold and hypoxia stress were studied, and underlying transcriptome mechanisms in the larva were pinpointed. The third instar was more tolerant than 12-h-old egg and 8-day-old pupa when treated at 3 °C + 1% O2 for 7 days, with 34.00% ± 5.22% larval survival. Hypoxia influenced the effect of cold treatment on D. suzukii. Larval survival decreased at 3 °C + 1% O2 , but increased at 0 °C + 1% O2 . Survival increased with temperature between 0 and 5 °C + 1% O2 , but decreased significantly at 25 °C + 1% O2 . RNA-sequencing results showed that the Tweedle (Twdl) family was upregulated and uniquely enriched in larvae treated at 3 °C + 1% O2 . In addition, RNA interference-mediated silencing of a key Twdl gene reduced the survival rate after cold and hypoxia treatment. Hypoxia was able to influence the effect of cold treatment on the survival of D. suzukii positively or negatively. Structural constituents of the chitin-based cuticle, in particular Twdl genes, body morphogenesis, and ATP synthesis-coupled proton transport were involved in the tolerance to cold and hypoxia. In future, the Twdl gene could be used as a nanocarrier delivering RNA pesticides to control D. suzukii in the field and so prevent its worldwide spread. © 2023 Society of Chemical Industry.

Abstract 1314: Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles

Abstract Triple negative breast cancer (TNBC) is the most lethal subtype of all breast cancers leading to high mortality rates. Tumor-derived extracellular vesicles (TEV) along with their cell-type specific cargo are greatly implicated in tissue-specific metastasis. In addition to metastasis, TEV play a crucial role in interacting with the distant microenvironment & shaping a pre-metastatic niche (PMN) for tumor cells to home. Understanding the mechanisms by which TEV promote PMN development has great potential to improve the diagnosis, prognosis, & treatment of TNBC. Annexin A2 (AnxA2) is a plasma & endosomal membrane-associated protein. High levels of AnxA2 in TNBC patients have been correlated with poor distant metastasis-free survival and poor overall survival. It is abundantly present in the TEV & recruits cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted TNBC-derived extracellular vesicles (EVs) led to reduced metastasis in lungs & brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in TNBC-derived EVs has been reported, its molecular role in the formation & development of PMN via EVs is still unexplored. We aim to evaluate the implications of AnxA2 in EVs at distant sites to mediate tumor cell homing & elucidate mechanisms promoting TNBC metastasis. We have employed RNA interference-mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. We observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. Differential ultracentrifugation was used to isolate EVs from cell culture supernatant and the physical characterization was done using Nanoparticle Tracking Analyzer. Biological characterization was done in concordance with MISEV 2018 guidelines. Using immunoblotting we confirmed reduced levels of AnxA2 in EVs derived from AnxA2 depleted TNBC cells. We verified their purity using EV enriched markers - ESCRT, Heat shock proteins & tetraspanins such as CD81, CD9, CD63 & confirmed the absence of negative markers - GM130 & cytochrome c. Interestingly, a reduced yield of EV was observed with AnxA2 depletion indicating a potential effect on EV biogenesis and release. Additionally, the EVs will be subjected to quantitative proteomic analysis to identify differentially expressed proteins upon loss of AnxA2. We will further carry out transcriptome profiling of the AnxA2 depleted TNBC cells and -derived EVs to identify the differentially expressed genes. The role of AnxA2 in TEV biogenesis, release and selective cargo loading will lead to potential identification and understanding of the novel secretory and EV protein that may act as a functional regulator in promoting advanced metastasis in TNBC. [This research is supported by the NCI under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha.] Citation Format: Rucha Trivedi, Jamboor K. Vishwanatha. Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1314.

RNA interference-mediated silencing of retinoid X receptor causes reproductive failure in the sea lice Caligus rogercresseyi

RNA interference-mediated silencing of retinoid X receptor causes reproductive failure in the sea lice Caligus rogercresseyi

EIF3 subunit M regulates blood meal digestion in Rhodnius prolixus affecting ecdysis, reproduction, and survival.

In triatomines, blood-feeding triggers many physiological processes including post embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define 2 different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited molting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines.

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation

BackgroundCanine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing.MethodsThis study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA.ResultsMalpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed.ConclusionsOur data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity.Graphical

Alfin-like transcription factor VqAL4 regulates a stilbene synthase to enhance powdery mildew resistance in grapevine.

Resveratrol is a phytoalexin that is synthesized by stilbene synthase (STS). Resveratrol in the human diet is known to have beneficial effects on health. We previously identified six novel STS (VqNSTS) transcripts from the transcriptome data of Vitis quinquangularis accession Danfeng-2. However, the functions of and defensive mechanisms triggered by these VqNSTS transcripts remain unknown. In the present study, we demonstrate that the expression of five of these six novel members, VqNSTS2-VqNSTS6, can be induced by the powdery mildew-causing fungus Uncinula necator. Additionally, overexpression of VqNSTS4 in the V. vinifera susceptible cultivar Thompson Seedless promoted accumulation of stilbenes and enhanced resistance to U. necator by activating salicylic acid (SA) signalling. Furthermore, our results indicate that the Alfin-like (AL) transcription factor VqAL4 can directly bind to the G-rich element (CACCTC) in the VqNSTS4 promoter and activate gene expression. Moreover, overexpression of VqAL4 in Thompson Seedless enhanced resistance to U. necator by promoting stilbene accumulation and activating SA signalling. Conversely, RNA interference-mediated silencing of VqNSTS4 and VqAL4 resulted in increased susceptibility to U. necator. Collectively, our results reveal that VqNSTS4, regulated by VqAL4, enhances grapevine resistance to powdery mildew by activating SA signalling. Our findings may be useful to improve disease resistance in perennial fruit trees.

Stabilized Double-Stranded RNA Strategy Improves Cotton Resistance to CBW (Anthonomus grandis).

Cotton is the most important crop for fiber production worldwide. However, the cotton boll weevil (CBW) is an insect pest that causes significant economic losses in infested areas. Current control methods are costly, inefficient, and environmentally hazardous. Herein, we generated transgenic cotton lines expressing double-stranded RNA (dsRNA) molecules to trigger RNA interference-mediated gene silencing in CBW. Thus, we targeted three essential genes coding for chitin synthase 2, vitellogenin, and ecdysis-triggering hormone receptor. The stability of expressed dsRNAs was improved by designing a structured RNA based on a viroid genome architecture. We transformed cotton embryos by inserting a promoter-driven expression cassette that overexpressed the dsRNA into flower buds. The transgenic cotton plants were characterized, and positive PCR transformed events were detected with an average heritability of 80%. Expression of dsRNAs was confirmed in floral buds by RT-qPCR, and the T1 cotton plant generation was challenged with fertilized CBW females. After 30 days, data showed high mortality (around 70%) in oviposited yolks. In adult insects fed on transgenic lines, chitin synthase II and vitellogenin showed reduced expression in larvae and adults, respectively. Developmental delays and abnormalities were also observed in these individuals. Our data remark on the potential of transgenic cotton based on a viroid-structured dsRNA to control CBW.

RNA interference-mediated silencing of ecdysone receptor (EcR) gene causes lethal and sublethal effects on melon aphid, Aphis gossypii

RNA interference-mediated silencing of ecdysone receptor (EcR) gene causes lethal and sublethal effects on melon aphid, Aphis gossypii

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Immune function analysis of LsSd, a transcription factor of the Hippo signaling pathway, in the cigarette beetle Lasioderma serricorne.

The Scalloped (Sd) is a transcription factor that regulates organ size control in the Hippo-signaling pathway. Recent studies have showed that Hippo signaling also functions in the innate immune response. Although the Sd gene has been reported in many insects, their immune functions remain unexplored. In this study, the LsSd gene of Lasioderma serricorne, with a complete open reading frame that encodes a protein composed of 402 amino acids was identified. LsSd was predominantly expressed in early pupae. Tissue-specific analyses revealed that the highest concentrations of LsSd were detected in the midgut and brain. At 1–24 h after Escherichia coli infection, LsSd expression increased substantially. However, LsSd expression was downregulated 3–12 h after Staphylococcus aureus infection. RNA interference-mediated silencing of the LsSd transcript resulted in deformed, considerably smaller, and degenerated wings. Meanwhile, LsCycE, LsDiap1, and LsVg, which are involved in cell proliferation and wing development, were drastically reduced when LsSd was depleted. In a survival assay, the LsSd knockdown considerably decreased the susceptibility to S. aureus, a gram-positive bacterium. In addition, knockdown of LsSd remarkably downregulated the transcription of LsCact in response to S. aureus stimulation, while upregulating the expression of five immune-related genes. Our results provide conclusive proof for the important roles of LsSd in the immune response of L. serricorne.