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.

Virus-Induced Cancers in Africa: Epidemiology and Carcinogenesis Mechanisms

The resurgence of infectious diseases on the African continent plays a major role in the increase in cancer occurrence. Whereas in developed countries the causes of occurrence of cancers are related mainly to non-infectious factors;cancers of infectious origin become a dramatic particularity in Africa. The proportion of virus-induced cancers may reach up to 75% of cancer cases in certain countries. Oncogenic viruses such as human papilloma virus (HPV), hepatitis viruses B and C, human herpes virus 8 and Epstein Barr virus in association with human immunodeficiency virus are the main viral etiologies of cancers in Africa, representing around 30% of cancers causes. Optimistically, 30% of cancers could be prevented in Africa. However, health burden prevails on the continent due to the weakness of health policy especially regarding preventive medicine, but also the limited technical facilities, poor manpower and insufficient political commitment. We felt urgent to review the state of the art of the question, and necessary to analyze and publicize the current epidemiological advances in oncogenic viruses and virus-induced cancers in Africa. Prevention implies understanding, which is compulsory to reverse the current trends and to potentially instate a control of virus-induced cancers.

Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS’, where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR, RhA G, and RhNUDXin rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.

Virus-induced gene silencing and its application in plant functional genomics

Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and reverse genetics to identify plant functional genes. Many viruses have been developed into virus-induced gene silencing vectors and gene functions involved in development, biotic and abiotic stresses, metabolism, and cellular signaling have been reported. In this review, we discuss the development and application of virus-induced gene silencing in plant functional genomics.

Virus-induced Gene Silencing in Eggplant(Solanum melongena)

Eggplant （Solanum melongena） is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing （VIGS） has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS （PHYTOENE DESATURASE） in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su （Sulfur）, CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

Virus-Induced Gene Silencing in the Culinary Ginger （Zingiber officinale）： An Effective Mechanism for Down-Regulating Gene Expression in Tropical Monocots

Virus-induced gene silencing （VIGS） has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress-related responses. VlGS is well established for studies of model systems and crops within the Solanaceae, Brassicaceae, Leguminaceae, and Poaceae, but only recently has been applied to plants residing outside these families. Here, we have demonstrated that barley stripe mosaic virus （BSMV） can infect two species within the Zingiberaceae, and that BSMV-VIGS can be applied to specifically down-regulate phytoene desaturase in the culinary ginger Zingiber officinale. These results suggest that extension of BSMV-VlGS to monocots other than cereals has the potential for directed genetic analyses of many important temperate and tropical crop species.

Applications of virus-induced gene silencing for identification of gene function in fruit

With the development of bioinformatics,it is easy to obtain information and data about thousands of genes,but the determi nation of the functions of these genes depends on methods for rapid and effective functi on al identification.Virus-induced gene sile ncing(VIGS)is a mature method of gene functional identification developed over the last 20 years,which has been widely used in many research fields involving many species.Fruit quality formation is a complex biological process,which is closely related to ripening.Here,we review the progress and contribution of VIGS to our understanding of fruit biology and its advantages and disadvantages in determining gene function.

Cotton ethylene response factor Gh ERF91 is involved in the defense against Verticillium dahliae

Verticillium dahliae causes significant losses in cotton production.To reveal the mechanism of the defense response to V.dahliae in cotton,transcriptomic analyses were performed using cotton cultivars M138(V.dahliae-resistant)and P2(V.dahliae-susceptible).The results revealed 11,076 and 6,640 differentially expressed genes(DEGs)in response to V.dahliae,respectively.The weighted gene co-expression network analysis of 4,633 transcription factors(TFs)indicated a“MEblue”module containing 654 TFs that strongly correlate with resistance to V.dahliae.Among these TFs,the ethylene response factor Ghi_A05G10166(GhERF91)was identified as a putative hub gene with a defense response against V.dahliae.A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V.dahliae exposure in cotton.This study provides fundamental transcriptome data and a putative causal gene(GhERF91)associated with resistance to V.dahliae,as well as genetic resources for breeding V.dahliae-resistant cotton.

Knockdown of the atypical protein kinase genes GhABC1K2-A05 and GhABC1K12-A07 make cotton more sensitive to salt and PEG stress

Activity of bc1 complex kinase(ABC1K)is an atypical protein kinase(aPK)that plays a crucial role in plant mitochondrial and plastid stress responses,but little is known about the responses of ABC1Ks to stress in cotton(Gossypium spp.).Here,we identified 40 ABC1Ks in upland cotton(Gossypium hirsutum L.)and found that the Gh ABC1Ks were unevenly distributed across 17 chromosomes.The GhABC1K family members included 35 paralogous gene pairs and were expanded by segmental duplication.The GhABC1K promoter sequences contained diverse cis-acting regulatory elements relevant to hormone or stress responses.The qRT-PCR results revealed that most Gh ABC1Ks were upregulated by exposure to different stresses.Gh ABC1K2-A05 and Gh ABC1K12-A07 expression levels were upregulated by at least three stress treatments.These genes were further functionally characterized by virus-induced gene silencing(VIGS).Compared with the controls,the Gh ABC1K2-A05-and Gh ABC1K12-A07-silenced cotton lines exhibited higher malondialdehyde(MDA)contents,lower catalase(CAT),peroxidase(POD)and superoxide dismutase(SOD)activities and reduced chlorophyll and soluble sugar contents under NaCl and PEG stress.In addition,the expression levels of six stress marker genes(Gh DREB2A,Gh SOS1,Gh CIPK6,Gh SOS2,Gh WRKY33,and Gh RD29A)were significantly downregulated after stress in the Gh ABC1K2-A05-and Gh ABC1K12-A07-silenced lines.The results indicate that knockdown of Gh ABC1K2-A05 and Gh ABC1K12-A07 make cotton more sensitive to salt and PEG stress.These findings can provide valuable information for intensive studies of Gh ABC1Ks in the responses and resistance of cotton to abiotic stresses.

A small knottin-like peptide negatively regulates in wheat to stripe rust resistance during early infection of wheat

Although Blufensins(Bln)have important functions in the response of plants to biotic stress the precise functioning of Bln in wheat remains largely unknown.Here we isolated a Bln gene(TaBln4)from Suwon 11 infected by Puccinia striiformis f.sp.tritici(Pst).Expression of TaBln4 increased in host plants at the early stage of infection with a virulent Pst race(CYR31)but was unchanged in response to infection by an avirulent race(CYR23).Transcription levels of TaBln4 were also regulated by hormone and abiotic stresses.Expression of TaBln4 in tobacco leaves suppressed Bax-induced programmed cell death.Knockdown of TaBln4 by virus-induced gene silencing inhibited colonization of race CYR31 by increasing the accumulation of H2O2 and formation of hypersensitive responses(HR).Transient overexpression of TaBln4 by a transient overexpression system(BSMV-VOX)increased the susceptibility of wheat to CYR31.Results from bimolecular fluorescence complementation and pull-down assays demonstrated that TaBLN4 interacted with calmodulin.Taken together,our results suggest that TaBln4 negatively regulates resistance in wheat to Pst in a reactive oxygen species(ROS)-and HR-dependent manner.

Cloning and Function Identification of a Phytoene Desaturase Gene from Eucommia ulmoides

The phytoene desaturase(PDS)encodes a crucial enzyme in the carotenoid biosynthesis pathway.Silencing or inhibiting PDS expression leads to the appearance of mottled,chlorosis,or albino leaves.In this study,the CDS sequence of EuPDS(Eucommia ulmoides Phytoene Desaturase)was first cloned and then PDS was silenced in Nicotiana benthamiana.Result showed the expression level of EuPDS in leaves was higher than that in the roots and stems.In N.benthamiana leaves,which were treated by Agrobacterium for 24 h,photo-bleaching was shown on the fresh leaves one week after injection and the transcript level of PDS was down-regulated during the period of emersion.This suggested that EuPDS could silence PDS of N.benthamiana,so as to cause the phenotype of leaf whitening.PDS is the main reporter gene involved in virus-induced gene silencing(VIGS).This study offered molecular evidence for identifying PDS gene involved in Carotenoid’s biosynthesis pathway and the regulation networks in E.ulmides.It also laid a useful foundation for study on leaf discoloration mechanism of other woody plants.

陆地棉NF-YA基因家族的全基因组鉴定与功能分析

【目的】通过对陆地棉NF-YA基因家族进行全基因组鉴定,分析其表达特性,鉴定其中与棉花开花相关的基因。【方法】利用生物信息学方法系统分析了其理化性质、基因结构、共线性、Ka/Ks、顺式作用元件和表达模式,实时荧光定量聚合酶链反应(Polymerase chain reaction,PCR)分析表达特征,采用病毒诱导基因沉默技术验证基因功能。【结果】陆地棉中鉴定到29个NF-YA基因家族成员,分为5个亚组,定位在18条染色体上;全基因组复制和片段复制是GhNF-YA基因家族扩张的主要动力;启动子区含有大量光反应的顺式作用元件。GhNF-YA基因家族基因在茎、叶中高表达;12个GhNF-YA基因在早熟品种鲁棉研19号和晚熟品种鲁棉研37号中均在第三至六叶时期表达量较高,并且大部分基因在品种间表达差异显著。沉默GhNF-YA18基因的鲁棉研37号植株比对照提前11 d现蕾,并且该基因在白化期和现蕾期的表达量均低于未沉默对照棉株。【结论】本研究对陆地棉NF-YA基因家族进行了鉴定和表达分析,为进一步研究棉花开花调控的分子机制奠定了基础。

EB病毒诱导基因3蛋白的生物学效应

EB病毒诱导基因3(epstein-barr virus-induced gene3,EBI3)是一种在EB病毒感染的B淋巴细胞中发现并命名的红细胞生成素受体样糖蛋白。它参与组成细胞因子IL-27和IL-35,在T细胞的增殖,Th1、Th2和Th17细胞以及NK细胞诱导分化过程中具有重要的免疫调节效应。同时EBI3作为CD4+CD25+Foxp3+调节性T细胞中Foxp3转录因子的下游靶位,可以促进调节性T细胞的增殖,并对效应性T细胞具有抑制功能。EBI3这些重要的免疫活性在自身免疫性疾病、感染性疾病、肿瘤以及变应性疾病等临床多种疾病的发生、发展中均具有重要的生物学效应。

A MADS-box gene is involved in soybean resistance to multiple Soybean mosaic virus strains

Soybean mosaic virus(SMV)is a member of the genus Potyvirus that extensively impairs global soybean production.The full-length coding sequence of the MADS-box transcription factor Gm CAL was cloned from the SMV-resistant soybean cultivar Kefeng 1.SMV-induced expression analysis indicated that Gm CAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2.Gm CAL was expressed at high levels in flowers and pods but at lower levels in leaves.The gene was localized to the nucleus by subcellular localization assay.Virus-induced gene silencing did not increase the accumulation of SMV in Gm CAL-silenced Kefeng 1 plants(with silencing efficiency~80%)after SC8 inoculation.Gm CAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression.SMV content decreased significantly in Gm CAL-overexpressing NN1138-2 plants after SMVSC3,SMV-SC7,and SMV-SC8 inoculation in comparison with a vector control,showing that overexpression of Gm CAL conferred broad-spectrum resistance to multiple SMV strains.These results confirm that Gm CAL,a key regulator but not a specific SC8 resistance gene(Rsc8),is a positive regulatory transcription factor involved in soybean resistance to SMV.

TaRAR1 is Required for Lr24-Mediated Wheat Leaf Rust Resistance

Virus-induced gene silencing （VIGS） offers a rapid and high throughout technology platform for the analysis of gene function in plants. The barley stripe mosaic virus （BSMV） VIGS system was optimized in studies silencing phytoene desaturase expression in wheat, and demonstrated that infection with BSMV construct carrying a 412 bp fragment of TaRAR1 caused conversion of incompatible to compatible interactions to Lr24-mediated resistance in wheat TcLr24 and cultivar 5R615 harboring Lr24 whereas infection with a control construct had no effect on resistance or susceptibility. RT- PCR analysis showed that BSMV-induced gene silencing could be detected at mRNA levels. These studies indicated that TaRAR1 was a required component for Lr24-mediated race-specific resistance and the BSMV-VIGS was a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.

Study of viruses co-infecting white clover(Trifolium repens)in China

Globally, white clover（Trifolium repens L.） is commonly infected by plant viruses. It is grown at gardens, roadsides, and public areas as ornamental plants in northern China. Some leaves present disease symptoms that are similar to those of virus infection. However, to our knowledge, no records are available from China regarding white clover（Trifolium repens L.） virus co-infection. To determine the viral species that infect white clover in China, plant samples with virus disease symptoms were collected and virion morphology and ultrastructure morphology of co-infected plants were observed by electron microscopy; viruses were detected by enzyme linked immunosorbent assay（ELISA） and reverse transcription PCR（RT-PCR）. Virus co-infection was studied by double antibody sandwich enzyme linked immunosorbent assay（DAS-ELISA） and real-time fluorescence quantitative PCR（RT-q PCR） after rub-inoculation of virus-free white clover planted in a growth chamber（25°C） with a photoperiod of 16 h, using single or several purified virions. Results showed that there were six types of symptoms, including those of shrinking mosaic, shrinking and macular mosaic, severe mottle mosaic, yellow macular mosaic, shrinkage chlorisis, and ring plaque and ring stria. The incidence rates for each symptom were 20.93, 1.48, 16.85, 59.07, 1.30, and 0.37%, respectively, based on the field investigation. Two types of viral pathogens were identified as Alfalfa mosaic virus（AMV） and White clover mosaic virus（WCMV）. In mesophyll cells, virus particles with bacilliform virions formed aggregates and linear virions were bundle shaped. The detection rate of AMV was 100% in white clover samples by DAS-ELISA and RT-PCR detection, whereas that of WCMV was 83.33%. The co-infection rate was 83.33%. The relative contents of AMV and WCMV were significantly increased by 5.897-and 3.515-fold upon co-infection, when compared to that with single virus infection. We observed larger starch particles and fewer or collapsed chloroplast grana in co-infected plants; in addition, vacuoles were twisted and smaller, compared to those of healthy plants. To our knowledge, this is the first report of co-infection by AMV and WCMV in white clover, which has caused severe mosaicism and ultrastructure lesions in co-infected plants in China.

Up-regulation of a homeodomain-leucine zipper gene HD-1 contributes to trichome initiation and development in cotton

Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T_1 locus.The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv.Hai7124 was interrupted by a long terminal repeat(LTR)retrotransposon,while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc.T586.Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family,which mainly regulated epidermis hair development in plants.Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation.Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton,while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development.This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton,but also provides a reference for exploring the different forms of trichome development in plants.

Hepatocellular carcinoma occurrence in DAA-treated hepatitis C virus patients:Correlated or incidental? A brief review

Hepatitis C virus(HCV) chronic infection induces liver fibrosis and cirrhosis but is also responsible for a significant portion of hepatocellular carcinoma(HCC) occurrence. Since it was recognized as a causative factor of chronic hepatitis,there have been multiple efforts towards viral eradication,leading to the first-generation HCV treatment that was based on interferon(IFN)-α and its analogs,mainly PEGylated interferon-α(PEG IFNα). Sustained virological response(SVR),defined as the absence of detectable RNA of HCV in blood serum for at least 24 wk after discontinuing the treatment,was accepted as a marker of viral clearance and was achieved in approximately one-half of patients treated with PEG IFNα regimens. Further research on the molecular biology of HCV gave rise to a new generation of drugs,the so-called direct antiviral agents(DAAs). DAA regimens,as implied by their name,interfere with the HCV genome or its products and have high SVR rates,over 90%,after just 12 wk of per os treatment. Although there are no questions about their efficacy or their universality,as they lack the contraindication for advanced liver disease that marks PEG IFNα,some reports of undesired oncologic outcomes after DAA treatment raised suspicions about possible interference of this treatment in HCC development. The purpose of the present review is to investigate the validity of these concerns based on recent clinical studies,summarize the mechanisms of action of DAAs and survey the updated data on HCV-induced liver carcinogenesis.

Contribution of IL-17 to Mouse Hepatitis Virus Strain 3-induced Acute Liver Failure

Recently,the Th17 cells and IL-17 have been shown to play a critical role in the immune-mediated liver injury in hepatitis B,while their functions in acute liver failure have not been well elucidated yet.In this study,we primarily investigated the role of IL-17 in the development of mouse hepatitis virus strain 3(MHV-3)-induced acute liver failure.IL-17 mRNA levels in liver tissue were quantified by using quantitative real-time polymerase chain reaction,and cytokine IL-17 levels in liver tissue and serum were determined by using ELISA in MHV-3-induced murine fulminant hepatitis model.The IL-17 expression levels on CD4 + T and CD8 + T cells were determined by using flow cytometry.The correlation between IL-17 level and liver injury was studied.Th17 associated cytokines were also investigated by intracellular staining.Our results showed that the IL-17 expression was significantly elevated in the liver and serum of BALB/cJ mice infected with MHV-3.Moreover,a time course study showed that the percentage of both IL-17-producing CD4 + T cells and IL-17-producing CD8 + T cells was increased remarkably in the liver starting from 48 h and peaked at 72 h post-infection.There was a close correlation between hepatic or serum IL-17 concentration and the severity of liver injury defined by ALT level,respectively.Th17 associated cytokines,IL-6,IL-21 and IL-22,were also increased significantly at 72 h post-infection.It was concluded that IL-17 may contribute to the pathogenesis of MHV-3-induced acute liver failure.

TaARPC5 is required for wheat defense signaling in response to infection by the stripe rust fungus

Numerous studies using a combination of confocal microscopic-and pharmacological-based approaches have demonstrated that the actin cytoskeleton dynamically responds to pathogen infection.Here,we observed that phalloidin treatment induced actin nucleation,resulting in enhanced resistance of wheat against the stripe rust pathogen Puccinia striiformis f.sp.tritici(Pst).To define the mechanism underpinning this process,we characterized a family of conserved actin-binding proteins,the actin related protein(ARP)family,which controls actin polymerization.Specifically,we identified and characterized a wheat ARPC gene(Ta ARPC5),which encodes a 136-amino acid protein containing a P16-Arc domain,the smallest subunit of the ARP2/3 complex.Ta ARPC5 m RNA accumulation was induced following the infection of plants with the avirulent Pst strain,and following the elicitation with flagellin(e.g.,flg22)as well.Subcellular localization analysis revealed that Ta ARPC5 is primarily localized to the cortical actin cytoskeleton,and its precise cellular localizations suggest the proximity to processes correlated with the actin-organelle interface.Upon treatment with virulent Pst,Ta ARPC5-knockdown plants exhibited a significant reduction in the expression of PTI-specific m RNAs.Conversely,we observed enhanced induction of reactive oxygen species(ROS)accumulation and a decrease in Ta CAT1 expression following infection with an incompatible Pst isolate.Together with yeast complementation assays,the current study demonstrates a role for Ta ARPC5 in resistance signaling in wheat against Pst infection by regulating the host actin cytoskeleton.