Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Host-Induced Gene Silencing of Effector AGLIP1 Enhanced Resistance of Rice to Rhizoctonia solani AG1-IA

Rice sheath blight, caused by Rhizoctonia solani AG1-IA, is a major disease in rice-growing areas worldwide. Effectors of phytopathogenic fungi play important roles during the infection process of fungal pathogens onto their host plants. However, the molecular mechanisms by which R. solani effectors regulate rice immunity are not well understood. Through prediction, 78 candidate effector molecules were identified. Using the tobacco rattle virus-host induced gene silencing(TRV-HIGS) system, 45 RNAi constructs of effector genes were infiltrated into Nicotiana benthamiana leaves. The results revealed that eight of these constructs resulted in a significant reduction in necrosis caused by infection with the AG1-IA strain GD-118. Additionally, stable rice transformants carrying the double-stranded RNA construct for one of the effector genes, AGLIP1, were generated to further verify the function of this gene. The suppression of the AGLIP1 gene increased the resistance of both N. benthamiana and rice against GD-118, and also affected the growth rate of GD-118, indicating that AGLIP1 is a key pathogenic factor. Small RNA sequencing showed that the HIGS vectors were processed into si RNAs within the plants and then translocated to the fungi, leading to the silencing of the target genes. As a result, AGLIP1 might be an excellent candidate for HIGS, thereby enhancing crop resistance against the pathogen and contributing to the control of R. solani infection.

Michel Serres and Posthuman Subjectivities:Silencing and Trans-lation to Explore Co-implications

The contribution aims to examine the co-implications between Serres’work and posthumanist ideas of body and subjectivity.This scope is pursued primarily through the methodological choice of a gerund,such as silencing,and the harnessing of its performative,processual,and relational value.Building on Serres’conception of silence as a dilation of the me the paper will follow Serresian anti-Cartesian reflections on the interchangeability of subject and object,his conception of the pre-positional body,and his thematization of the soul-body relationship.In close inter-implication with the employment of silencing is then the choice,again as a methodological device,of the preposition trans,made to act in order to explore the affinities/overlaps/assonances between Serres’theorization of the body,dimension of the human,and posthumanist conceptions of body/subjectivity.

Stress response silencing by an E3 ligase mutated in neurodegeneration

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis,yet their prolonged activation induces apoptosis and disrupts organismal health1-3.How stress responses are turned off at the right time and place remains poorly understood.Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.

Features,Mechanisms and Applies of Post-transcriptional Gene Silencing in Transgenic Plants

Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.

Using Gene Silencing Technology to Create Blast Resistant Rice Resources

Rice blast disease is one of the most devastating diseases in rice production,which severely affects the high and stable yield of rice.The formation of appressorium plays a key role in the pathogenesis of Magnaporthe grisea in rice.It has been confirmed that a P-type ATPase （P-ATPase） is involved in the formation of appressorium.A number of small molecular substances are able to enter the pathogen from the host during the interactions between pathogens and hosts,thus resisting the infection of pathogens.In this study,a 232 bp DNA sequence with good specificity from the first exon of P-ATPase gene MgAPT2 was used as an interference fragment and was inserted into interference vector forward and reversely.The interfering vector was then transformed into rice blast-susceptible rice variety Nipponbare via Agrobacterium-mediated transformation.Identification of rice plants inoculated with M.grisea at the seedling stage and detection of the expression level of P-ATPase gene MgAPT2 showed that the expression level of MgAPT2 gene in transgenic plants was reduced and the rice blast resistance was improved.This study provided a new way for the innovation of rice germplasm resources resistant to rice blast disease.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content,Alters Transcripts of Phytic Acid Biosynthetic Genes,and Modulates Mineral Distribution in Rice Seeds

Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development.However,it is known as an anti-nutrient compound owing to its potent chelating property.Thus,reducing the phytic acid content in crops is desirable.Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier.However,the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated.In this study,tissue specific RNA interference(RNAi)-mediated down-regulation of a major ITPK homolog(OsITP5/6K-1)resulted in 46.2%decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content.Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes,without significantly affecting the transcript levels of other OsITPK homologs.Furthermore,the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds.Additionally,low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm,respectively.Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds.Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth.The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.

Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Virtual design and performance prediction of a silencing air cleaner used in an I.C.engine intake system

This paper reports results of the authors’ studies on the virtual design method used in the development of low noise intake system of I.C. engine. The resulting high pass-by noise at level above the legislative target at full throttle when engine speed was around 5200 r/min necessitated a BEM-aided redesign task, following the typical process of design and development of an intake system. During the initial design, based on the acoustic theory and the requirements (1. The air flux of the redesigned should equal to or exceed the value of the original flux; 2. The filtering area must not be degraded), and considering the constraint of space in the engine compartment, total volume and rough internal dimensions were determined. During the detailed design, the exact internal dimensions of the air cleaner were determined, and an effective method was applied to improve the acoustic performance at low frequency. The predicted sound power of the intake system indicated that the objective of reducing the overall engine noise by minimizing intake system noise was achieved.

Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach

Global prophylactic vaccination programmes have helped to curb new hepatitis B virus(HBV)infections.However,it is estimated that nearly 300 million people are chronically infected and have a high risk of developing hepatocellular carcinoma.As such,HBV remains a serious health priority and the development of novel curative therapeutics is urgently needed.Chronic HBV infection has been attributed to the persistence of the covalently closed circular DNA(cccDNA)which establishes itself as a minichromosome in the nucleus of hepatocytes.As the viral transcription intermediate,the cccDNA is responsible for producing new virions and perpetuating infection.HBV is dependent on various host factors for cccDNA formation and the minichromosome is amenable to epigenetic modifications.Two HBV proteins,X(HBx)and core(HBc)promote viral replication by modulating the cccDNA epigenome and regulating host cell responses.This includes viral and host gene expression,chromatin remodeling,DNA methylation,the antiviral immune response,apoptosis,and ubiquitination.Elimination of the cccDNA minichromosome would result in a sterilizing cure;however,this may be difficult to achieve.Epigenetic therapies could permanently silence the cccDNA minichromosome and promote a functional cure.This review explores the cccDNA epigenome,how host and viral factors influence transcription,and the recent epigenetic therapies and epigenome engineering approaches that have been described.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Gene silencing:Double-stranded RNA mediated mRNA degradation and gene inactivation

The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.

Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cellular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffusion-weighted imaging（DWI）. We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and si RNA-aquaporin-4 was immediately injected via the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefficient value on T2-weighted imaging（T2WI） and DWI gradually increased within 0.5–6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2 WI and DWI reduced, relative apparent diffusion coefficient value was increased, and cellular edema was obviously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefficient value was similar between treatment and model groups, but angioedema was still obvious in the treatment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefficient value and the area of high signal intensity on T2 WI and DWI can reflect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

Effects of gene silencing of CypB on gastric cancer cells

Objective:To determine the effect of gene silencing of cyclophilin B(CypB)on growth and proliferation of gastric cancer cells.Methods:CypB siRNA lentivirus(LV-CypB-si)and control lentivirus(LV-si-con)were produced.CypB expression in gastric cancer cell lines was detected by Western blot.BGC823 and SGC7901 cells were chosen to be infected with LV-sicon and LV-CypB-si,and stable transfectants were isolated.The cell groups transfected with LV-CypB-siRNA,LV-siRNA-con and transfected no carrier were served as the experimental group,the implicit control group and the blank control group respectively.MTT and colony formation assays were used to examine the effect of CypB on the cell growth and proliferation in vitro.Cell cycle was analyzed with flow cytometry.The expression of VEGFR of BGC823-si and SGC7901-si was detected by Western blot.Results:Gene silencing of CypB can inhibit gastric cancer cell growth,proliferation,cell cycle progress and tumorigenesis.CypB expression level was obviously higher in SGC7901 and BGC823 than MKN28 and GES.These two cell lines were infected with LV-si-con and LV-CypB-si respectively.MTT and cloney formation assays showed a significantly decreased rate of cell proliferation from the forth day or the fifth day in cells transfected with LV-CypB-si(P<0.05).Down-regulation of CypB resulted in slightly decreased percentage of S phase and increased percentage of G_1(P<0.05).These findings indicated that CypB could promote the G_1-S transition of gastric cancer cell.In addition,the expression of VEGF of BGC823 and SGC7901 transfected with CypB siRNA was reduced in comparison with the implicit control group and the blank control group.Conclusions:Gene silencing of CypB decreases gastric cancer cells proliferation and in vivo tumorigenesis.These findings indiccate CypB could be a potential biomarker and therapeutic target for gastric cancer.

Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer

AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.

Involvement of methylation-associated silencing of formin 2 in colorectal carcinogenesis

AIM To investigate whether promoter methylation is re-sponsible for the silencing of formin 2 （ FMN2） in colorectal cancer （CRC） and to analyze the association between FMN2 methylation and CRC.METHODSWe frst identifed the expression levels and methylation levels of FMN2 in large-scale human CRC expression data-sets, including GEO and TCGA, and analyzed the relation-ship between the expression and methylation levels. Then, the methylation levels in four CpG regions adjacent to the FMN2 promoter were assessed by MethylTarget？ assays in CRC cells and in paired colorectal tumor samples and adjacent nontumor tissue samples. Furthermore, we inhibited DNA methylation in CRC cells with 5-Aza-2’-deoxycytidine and assessed the expression of FMN2 by qRT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.RESULTSA statistically significant downregulation of FMN2 ex-pression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples （AUC = 0.8432, P 〈 0.0001）. MethylTarget？ assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was signifcantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, the highest methylation of FMN2 occurred in tissues from cases of early-stage CRC, including cases with no regional lymph node metastasis （N0）, cases in stages Ⅰ and Ⅱ, and cases with no lymphovascular inva-sion, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hyper-methylation was more common in patients 〉 60 years old and in colon cancer tissue.CONCLUSIONFMN2 promoter hypermethylation may be an important early event in CRC, most likely playing a critical role in cancer initiation, and can serve as an ideal diagnostic biomarker in elderly patients with early-stage colon cancer.

Studies on Trans-Generational Transcriptional Silencing of <i>cry</i>1<i>Ac</i>Gene in Tobacco Transgenics

Developing transgenics that express high levels of Cry1Ac protein, and at the same time, are phenotypically normal, has not been an easy task to achieve. It has been routinely observed that most of the transgenic plants that survive, show no or extremely low levels of Cry1Ac protein. However, all of these plants do express the selectable marker, nptII gene. In the present study, we record an interesting observation of how one of the genes (cry1Ac) on a single T-DNA fragment is selectively silenced, keeping the expression of the other gene (nptII) intact. Further, this silenced state is inherited.

Michel Serres and the Posthumanism: Silencing, Recognizing, and Working on Absences

Faced with a socio-political-mediatic arena that continues to return the ballet of pandemic,climate change,fourth industrial revolution,sixth mass extinction,war etc.,the reflection of Michel Serres and Posthumanism put forth instances for silencing of the anthropocentric logos,and for recognition of the multiplicity,variety,possibility of things and of the human in co-belonging with them,as well as instances for working on these same multiplicities,varieties,possibilities,that are often absences,black holes,repressed of philosophical thought.