Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA beta complexes

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA beta to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus-DNA beta complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus-DNA beta complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings.     

The Red clover necrotic mosaic virus RNA-2 encoded movement protein is a second suppressor of RNA silencing

The replication complex of Red clover necrotic mosaic virus (RCNMV) has been shown to possess silencing suppression activity. Here a newly developed viral-based assay for the identification of silencing suppression activity was used to provide evidence for a second, mechanistically distinct method of silencing suppression provided for by the RCNMV movement protein (MP). This new assay relies on Turnip crinkle virus with its capsid protein replaced with green fluorescent protein to act as a reporter (TCV-sGFP). In the presence of a protein with silencing suppression activity TCV-sGFP readily moves from cell-to-cell, but in the absence of such a protein TCV-sGFP is confined to small foci of infection. This TCV-sGFP assay was used to identify MP as a suppressor of RNA silencing, to delimit essential amino acids for this activity and uncouple silencing and movement functions.     

Enhanced viral pathogenesis associated with a virulent mutant virus or a virulent satellite RNA correlates with reduced virion accumulation and abundance of free coat protein

SatC, a chimeric satellite RNA associated with Turnip crinkle virus (TCV), intensifies the symptoms of TCV on all symptomatic hosts yet attenuates the symptoms of a TCV mutant that expresses low levels of a defective coat protein (CP). We now report that TCV virion levels were substantially reduced by the presence of satC or when two amino acids were inserted at the N-terminus of the CP, resulting in similarly enhanced symptoms. Since the TCV CP is a suppressor of RNA silencing, increased levels of resultant free CP could augment silencing suppression, resulting in enhanced colonization of the plant.     

The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens

Helper component-protease (HC-Pro) is a plant viral suppressor of RNA silencing, and transgenic tobacco expressing HC-Pro has increased susceptibility to a broad range of viral pathogens. Here we report that these plants also exhibit enhanced resistance to unrelated heterologous pathogens. Tobacco mosaic virus (TMV) infection of HC-Pro-expressing plants carrying the N resistance gene results in fewer and smaller lesions compared to controls without HC-Pro. The resistance to TMV is compromised but not eliminated by expression of nahG, which prevents accumulation of salicylic acid (SA), an important defense signaling molecule. HC-Pro-expressing plants are also more resistant to tomato black ring nepovirus (TBRV) and to the oomycete Peronospora tabacina. Enhanced TBRV resistance is SA-independent, whereas the response to P. tabacina is associated with early induction of markers characteristic of SA-dependent defense. Thus, a plant viral suppressor of RNA silencing enhances resistance to multiple pathogens via both SA-dependent and SA-independent mechanisms.     

神经元素3基因沉默对脐源性胰岛前体细胞MafA表达的影响

目的 探讨神经元素3（Ngn3）基因在人脐带间充质干细胞（hUMSCs）向胰岛前体细胞分化过程中的作用以及对肌腱膜纤维肿瘤基因同系物A（MafA）表达的影响。方法 组织块法分离培养并鉴定hUMSCs;采用分阶段联合诱导法诱导hUMSCs向胰岛前体细胞分化,将其分为正常诱导组、基因沉默组、空病毒转染组,后两组在诱导第7天分别转染干扰病毒和空病毒,嘌呤霉素筛选后检测感染效果;21 d诱导结束后倒置相差显微镜和电子显微镜观察细胞形态变化;免疫细胞化学技术检测诱导后各组细胞胰岛素、胰高血糖素、Ngn3的表达;Real-time PCR、Western blotting检测Ngn3以及MafA的表达变化。结果 成功分离培养hUMSCs;分阶段联合诱导使正常诱导组和空病毒转染组细胞分化为胰岛前体细胞,Ngn3、胰岛素、胰高血糖素呈阳性表达,电子显微镜观察可见分泌颗粒;成功沉默了基因沉默组细胞的Ngn3基因,且该组细胞Ngn3、胰岛素、胰高血糖素呈阴性表达;Ngn3、MafA在基因沉默组明显下降（P<0.05）。结论 Ngn3基因沉默阻滞了hUMSCs向胰岛前体细胞的分化,并抑制了MafA的表达。     

沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响

目的 探讨beclin1基因对缺氧缺糖/复氧复糖小鼠海马神经元细胞系HT22细胞凋亡的影响。方法 取对数生长期的HT22细胞,随机分为4组：正常组（normal）、缺氧缺糖/复氧复糖（OGD/R）模型组（model）、beclin1基因沉默组（beclin1^-/-）、转染对照组（control）。除normal组外,其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。利用RNAi技术,针对小鼠cDNA序列设计beclin1干扰序列,用脂质体Lipo2000包裹后转染至HT22细胞。于转染48 h后用荧光显微镜观察转染效率,Western blotting检测细胞beclin1 表达情况。各组细胞均于复氧复糖24 h后采用CCK-8法检测细胞活力,乳酸脱氢酶（LDH）法检测细胞损伤情况,免疫荧光染色检测Bax、Bcl-2表达及比值变化,Western blotting检测LC3、P62及Caspase-3表达。SPSS 19.0 统计学软件进行数据分析。结果 与normal组相比,model组细胞活力及P62蛋白表达显著降低（P<0.01）,乳酸脱氢酶（LDH）漏出率、LC3Ⅱ/LC3Ⅰ、Caspase-3表达及Bax/Bcl-2均显著升高（P<0.01）。与model组相比,beclin1^-/-组细胞活力及LC3Ⅱ/LC3Ⅰ 表达显著降低（P<0.01）,LDH漏出率、Bax/Bcl-2及P62、Caspase-3表达显著升高（P<0.01）;control组与model组比较差异无显著性。结论 沉默beclin1抑制细胞自噬可使缺氧缺糖/复氧复糖处理的HT22细胞损伤加重,细胞凋亡进一步增加。     

慢病毒介导的GOLPH3基因沉默对人胃癌SGC-7901细胞增殖、侵袭和迁移能力的影响*

 目的：通过体外实验探讨沉默高尔基体磷蛋白3（Golgi phosphoprotein 3,GOLPH3）基因对胃癌SGC-7901细胞增殖、侵袭和迁移能力的影响。方法：将携带GOLPH3 shRNA的慢病毒载体转染SGC-7901细胞,建立稳定沉默GOLPH3的细胞株LV-GOLPH3-RNAi,分别用实时荧光定量PCR和Western blotting技术检测GOLPH3 mRNA 和蛋白的表达。MTT法检测细胞增殖力,Transwell迁移和侵袭实验分别检测细胞的迁移力和侵袭力。结果：稳定沉默GOLPH3的细胞株构建成功;GOLPH3 mRNA 和蛋白表达水平明显降低（P<0.05）。与scrambled组和空白对照组相比,转染后LV-GOLPH3-RNAi组细胞的增殖力受到抑制（P<0.05）,迁移力(56.7±1.5 vs 186.0±3.4、183.3±4.2,P<0.05） 和侵袭力 （33.5±3.0 vs 85.0±3.9、83.1±4.4,P<0.05） 也明显受到抑制。结论：沉默GOLPH3基因的表达可抑制人胃癌SGC-7901细胞的增殖力、迁移力和侵袭力,提示GOLPH3有可能成为潜在的肿瘤标志物和独立的预后因子。     

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato.     

Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNAβ satellite

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of geminiviruses. Capsid proteins (CPs) of geminiviruses are multifunctional proteins thought to be involved in this process. The CP of monopartite geminiviruses is absolutely essential for virus movement. To more precisely examine the role of CP, we have constructed a series of single and double deletions into the coding sequence of Tomato leaf curl Java virus (ToLCJAV) CP and examined sub-cellular localization using transient expression of GFP fusion proteins. In this report, the domains of the CP encoded by ToLCJAV localized in the nucleus/nucleolus and cytoplasm in transfected cells were mapped. Deletion analysis revealed that the Arg-rich cluster from amino acids (aa) ¹⁶KVRRR²⁰ in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). The region from aa ⁵²RKPR⁵⁵ contained basic amino acid cluster was capable to redirect the CP to the nucleus. Further, both transient expression and yeast hybrid assays demonstrated that CP was capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa (²⁴⁵LKIRIY²⁵⁰) reside at C-terminal part of CP. This hydrophobic region caused transport of GFP to the cytoplasm. However, ToLCJAV CP NLSs and NES show peculiarities in the number and position of basic residues. Taken together, these results demonstrated that ToLCJAV CP shuttles between the nucleus and cytoplasm, such an activity homolog to bipartite geminivirus BV1 ORF.     

Characterization of Rhynchosia yellow mosaic Yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico

Rhynchosia minima (L.) DC. (Fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, México [1]. To characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. Sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic R. minima, and the name Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) is proposed. Pairwise comparisons indicated that RhYMYuV DNA-A [2,597 nt, (EU021216)] and DNA-B [2,542 nt, (FJ792608)] components shared the highest nt sequence identity with Cabbage leaf curl virus (CaLCuV), 87% for component A and 71% for component B. Phylogenetic analysis indicated that both components of RhYMYuV are most closely related to other New World begomoviruses, having as closest relatives immediate outliers to the major Squash leaf curl virus (SLCV) clade. Recombination analysis of the RhYMYuV genome indicated that the DNA-A component has arisen through intermolecular recombination. R. minima plants inoculated with the monomeric clones developed a bright yellow mosaic similar to symptoms observed in naturally infected plants, confirming that the clones were infectious. Nicotiana benthamiana plants biolistically inoculated with monomeric clones developed curling and chlorosis in the newly emerging leaves. RhYMYuV was also detected in symptomatic Desmodium sect. Scorpiurus Benth. (Fabaceae) that were collected near the RhYMYuV-infected plants.     

Adiponectin gene ADIPOQ SNP associations with serum adiponectin in two female populations and effects of SNPs on promoter activity

Adiponectin is an insulin sensitiser in muscle and liver, and low serum levels characterise obesity and insulin resistance. Eight tagging single nucleotide polymorphisms (tSNPs) in the ADIPOQ gene and promoter were selected, and association with serum adiponectin was tested, in two independent samples of Caucasian women: the Chingford Study (n = 808, mean age 62.8 +/- 5.9 years) and Twins UK (n = 2,718, mean age 47.4 +/- 12.6 years). In the Chingford cohort, -11391 G/A, -10066 G/A (rs182052), -7734 C/A (rs16861209), +276 G/T (rs1501299) and +3228 C/T (rs1063537) were significantly associated with fasting serum adiponectin (Ps = 1.00 x 10(-4) to 1.40 x 10(-2)). Associations with all except +3228 C/T were replicated in the Twins UK cohort (Ps = 3.19 x 10(-9) to 6.00 x 10(-3)). In Chingford subjects, the 12 most common 8-SNP haplotypes (frequency 1.90%) explained 2.85% (p = 5.00 x 10(-2)) and in Twins UK subjects, the four most common 5-SNP haplotypes (frequency > 5.00%) explained 1.66% of the variance (p = 5.83 x 10(-7)). To investigate effects of -11391 G/A (rs17300539) and -11377 C/G (rs266729) on promoter activity, 1.2 kb of the ADIPOQ promoter region was cloned in a luciferase reporter plasmid, and the four haplotypes were transfected in differentiated 3T3-L1 adipocytes. No significant allelic effects on promoter activity were found.     

SPA基因启动子的克隆及转录靶向活性分析

目的：克隆大鼠肺表面活性蛋白A（SPA）基因启动子并构建该启动子的荧光报告系统,探讨该启动子的活性及转录靶向性，为进一步研究SPA 基因的表达调控机制和探讨靶向性基因治疗奠定基础。方法:①从GenBank中获取大鼠SPA 基因序列，对其上游基因组序列进行计算机生物信息学分析，推断其上游序列约163bp的区域具有启动子功能。②利用PCR技术扩增SPA 基因上游启动子序列,将其亚克隆入pGL3-basic 中,构建pGL3-SPA 质粒，将其亚克隆于pGL3-control 中，构建pGL3-SPA-enhancer 质粒。③将构建pGL3-SPA质粒、pGL3-SPA-enhancer 质粒、pGL3-control质粒和pGL3-basic 质粒分别与内参质粒pRL-TK共转染入A549细胞和H441细胞, 用双荧光素酶报告系统检测该质粒在两种细胞中的荧光素酶活性表达。 结果:酶切及测序结果均证实成功克隆了SPA基因启动子序列,并已将该序列正确插入到荧光素酶报告基因系列载体中。重组的pGL3-SPA-enhancer 质粒、pGL3-SPA质粒转染H441 细胞后可以检测到荧光素酶的高表达。结论:成功构建了含有SPA 基因启动子序列的荧光素酶基因报告系统,证实了它在高表达SPA蛋白的细胞中有较高的转录活性， 为下一步研究SPA 基因功能及其转录调控以及探讨基因治疗的靶向性奠定了基础。