Hypovirulence and Double-Stranded RNA in Botrytis cinerea

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.     

Hypovirulence and Double-Stranded RNA in Sclerotinia homoeocarpa

ABSTRACT One hundred and thirty-two isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass, were evaluated for virulence on swards and detached leaves of creeping bentgrass and for the presence of double-stranded RNA (dsRNA). In at least four isolates, the hypovirulent phenotype was associated with the presence of specific segments of dsRNA. In addition, these hypovirulent isolates often grew slowly on potato dextrose agar (PDA), formed thin colonies with atypical colony margins, and failed to produce typical black stroma. The hypovirulent phenotype and dsRNA were transmitted from hypovirulent isolate Sh12B to virulent isolate Sh48B, and the converted isolate was hypovirulent and contained dsRNA. The hypovirulent phenotype and dsRNA also were transmitted to at least four other isolates of the pathogen, including the fungicide-resistant, dsRNA isolate KY-7. Converted isolates of KY-7 developed the hypovirulent phenotype, grew on fungicide-amended medium, and contained dsRNA. Subcultures of hypovirulent isolate Sh12B that did not contain dsRNA were obtained through curative treatment using cycloheximide-containing medium and heat. Cured subcultures grew faster on PDA, had more typical colony morphologies, were more virulent on bentgrass leaves, and did not contain dsRNA. No cured subcultures were obtained from hypovirulent isolate Sh09B. Isolates regenerated from protoplasts of hypovirulent isolate Sh12B were not cured, remained hypovirulent, and contained dsRNA. Transmission of hypovirulence and dsRNA in S. homoeocarpa has potential as a novel approach to the management of dollar spot of turfgrass.     

卫星RNA作为黄瓜花叶病毒的生防因子——Ⅲ.含有卫星RNA的黄瓜花叶病毒S51的某些生物学特性的研究

 1982~1983年比较了含有卫星RNA的黄瓜花叶病毒(CMV)S51和CMV分离物的寄主范围、症状反应和蚜虫传染的特性。对属于8科25种以蔬菜为主的植物的接种和回接实验证明:S51比CMV的寄主范围窄得多,只侵染茄科、葫芦科和藜科中的10种植物,CMV侵染6科的20种植物。S51除在西葫芦的部分植株上有花叶症状外,均无症状;而CMV能引起重花叶、畸形和坏死等症状。桃蚜对S51的传染效率比CMV低,潜育期延长2~6天。     

小麦黄花叶病毒RNA1基因组序列分析及RNA2 28kDa蛋白的表达

 以我国河南潢川感染黄花叶病的小麦为材料,提纯小麦黄花叶病毒,抽提RNA。以小麦黄花叶病毒总RNA为模板,采用引物延伸法,通过3次反转录,获得了分别包括小麦黄花叶病毒RNA1大部分编码区及5'末端非编码区的多种cDNA产物。     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Rapid Identification of Clavibacter michiganensis Subspecies Sepedonicus based on the Stable Low Molecular Weight RNA (LMW RNA) Profiles

Clavibacter michiganensis subsp. sepedonicus causes potato ring rot disease. The identification process for this bacterium is complex and long. This work demonstrates that the stable low-molecular-weight (LMW) RNA profiles allow their rapid identification. Staircase electrophoresis in polyacrylamide gels was used to analyze the LMW RNA profiles of 54 strains of C. michiganensis subsp. sepedonicus from different geographic origins. The profiles of several strains of other subspecies of C. michiganensis and other pathogens of potatoes were also analyzed. All the strains of C. michiganensis subsp. sepedonicus had the same LMW RNA profile. They had a band in class 2 of tRNA that was absent in the other subspecies of the species C. michiganensis. Also, the LMW RNA of C. michiganensis subsp. sepedonicus was different with respect to the LMW RNA profiles of other pathogens of potato. The results indicate the possible utilization of LMW RNA profiles in identification of the bacteria causing potato ring rot disease.     

寄生蜂RNA病毒研究进展

本文概述了寄生蜂体内RNA病毒的研究进展。迄今,在寄生蜂中已鉴定获得17种RNA病毒,分属7个科、3种基因组型,其中冠状病毒科、传染性软腐病病毒科、双顺反子病毒科和杯状病毒科等属正义单链RNA病毒;弹状病毒科和尼亚玛尼病毒科等属负义单链RNA病毒;呼肠孤病毒科属分段双链RNA病毒等。就不同寄生蜂RNA病毒的形态与基因组特征、传播途径以及与寄生蜂及其寄主间的相互关系作了比较,并就RNA病毒在寄生蜂成功寄生中的作用以及对寄生蜂的致病性等作了探讨,旨在为促进寄主-寄生蜂-病毒三者互作机制研究以及用于害虫协同防控提供参考。     

烟草丛顶病毒RNA依赖RNA聚合酶(RdRp)介导的体外复制体系

通过重叠PCR扩增得到烟草丛顶病毒(Tobacco bushy top virus,TBTV)中国分离物RdRp的编码序列,构建以pMALC2X为基础载体的原核表达载体pMAL-TB-RdRp。0.5 mM IPTG诱导可特异性表达分子量约为120 kDa的MBP-RdRp融合蛋白。温度梯度实验显示,18℃下诱导表达的MBP-RdRp融合蛋白的可溶性比例较高,约17%;经亲和层析纯化的MBPRdRp可特异性识别TBTV正链和负链的3'末端序列,催化体外复制;对正负链的3'末端的体外复制效率存在差别,识别负链3'末端的体外复制效率明显高于正链3'末端。本研究创建的TBTV RdRp介导的体外复制体系为进一步研究TBTV基因组复制调控奠定了基础。     

Association of double-stranded RNA with whitefly-mediated silvering in squash

A disorder of unknown aetiology appearing as silvering on squash (Cucurbita pepo) leaves has become prevalent in south Florida. Typical symptoms, observed after 2 weeks of exposure to adults of Bemisia tabaci , the sweet potato whitefly (SPWF) include vein clearing followed by silvering of whole leaves. Fruits from affected plants are blanched or streaked longitudinally. The silverleaf syndrome was not mechanically transmissible when crude extracts of symptomatic leaves were used as inoculum. Two sizes of double-stranded RNA (dsRNA) of c. 4.6 and 4.2 kilobase pairs (kbp) were consistently observed in leaf extracts of caged, whitefly-infested, symptomatic plants. In contrast, caged plants not exposed to whiteflies showed no symptoms and contained no detectable dsRN A. In addition, squash plants exposed to SPWF colonies from California neither developed silverleaf symptoms nor contained any dsRNA. Double-stranded RNA could also be readily detected in whitefly-infested yellow summer squash and zucchini from the field. However, asymptomatic eggplant and watermelon infested with SPWF from nearby fields did not contain detectable dsRNA. Dot-spot hybridization assays using an RNA-specific probe detected homologous sequences in both adults and nymphs of SPWF that induced silverleaf symptoms but not in adults and nymphs of SPWF that could not induce silverleaf symptoms. These results, along with the non-endogenous nature of the detected dsRNA suggest that the dsRNA is or is associated with the causal agent of whitefly-mediated leaf silvering in squash.     

Molecular mechanisms influencing efficiency of RNA interference in insects

RNA interference (RNAi) is an endogenous, sequence‐specific gene‐silencing mechanism elicited by small RNA molecules. RNAi is a powerful reverse genetic tool, and is currently being utilized for managing insects and viruses. Widespread implementation of RNAi‐based pest management strategies is currently hindered by inefficient and highly variable results when different insect species, strains, developmental stages, tissues, and genes are targeted. Mechanistic studies have shown that double‐stranded ribonucleases (dsRNases), endosomal entrapment, deficient function of the core machinery, and inadequate immune stimulation contribute to limited RNAi efficiency. However, a comprehensive understanding of the molecular mechanisms limiting RNAi efficiency remains elusive. Recent advances in dsRNA stability in physiological tissues, dsRNA internalization into cells, the composition and function of the core RNAi machinery, as well as small‐interfering RNA/double‐stranded RNA amplification and spreading mechanisms are reviewed to establish a global understanding of the obstacles impeding wider understanding of RNAi mechanisms in insects. © 2018 Society of Chemical Industry     

麦长管蚜OBP7基因的RNA干扰

OBP是蚜虫嗅觉功能中的一类重要分泌蛋白,能选择性地结合气味分子并进行信号转导。本试验使用显微注射法将麦长管蚜OBP7的小干扰RNA(siRNA)导入麦长管蚜体内,通过qRT-PCR检测OBP7mRNA相对表达量的变化情况。结果发现在siRNA浓度0.32μg/μL,注射量23nL,注射后24h,麦长管蚜成蚜的mRNA相对表达量降低到37.1%,证明了显微注射方式进行RNA干扰的可行性。在注射23nL的siRNA-467,经过36h后,麦长管蚜出现了对EβF趋性行为的改变(P0.05),表明OBP7蛋白在麦蚜对EβF的识别过程中具有重要作用。     

siRNA对烟草原生质体中TMV RNA积累的干扰作用研究(英文)

 RNA干扰被认为是转录后基因沉默的一种机制。RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达。本文以烟草花叶病毒126kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用。ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量。在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生。这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用。因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用。     

Association of three RNA molecules with potato mop-top virus

Summary Potato mop-top virus particles, purified from systemically infectedNicotiana benthamiana plants and then disrupted by heating with sodium dodecyl sulphate and 2-mercaptoethanol, contained only a single polypeptide of M_r 19 100 detectable by polyacrylamide gel electrophoresis. Single-stranded RNA preparations from virus particles, when subjected to electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturant, were shown to contain approximately equal proportions of three RNAs of 6.5, 3.2 and 2.5 kb. Double-stranded RNA preparations extracted from systemically infectedN. benthamiana leaves or from locally infectedN. debneyi leaves, was shown by polyacrylamide gel electrophoresis to contain a major species of 3.2 kbp and two minor species of 6.5 and 2.4 kbp.     

雀麦花叶病毒核酸特性的研究

 对用两相酚法提取的雀麦花叶病毒E株系和G株系的RNAs进行了聚丙烯酰胺凝胶电泳,其结果进一步证明RNA3a是BMVG株系中的一个新RNA组份。在昆诺藜上进行核酸侵染性测定,也再次证明其核酸的侵染需要RNA₁、RNA₂、RNA₃。而RNA3a和RNA₄对其症状表现也有一定的强作用。分别用BMV-E-RNAs和RNA3a做模板,合成其互补DNA（C-DNA）.采用NorthernBlot杂交分析法分别和E株系、G株系的RNA组份进行分子杂交实验,发现RNA3a和RNA3的核苷酸序列有同源性.这说明RNA3a属于RNA₃的亚基组份.而不是原来推测的卫星RNA.