A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

利用小RNA高通量测序技术检测玉米病毒

正玉米是我国重要的粮食作物,种植范围日趋增大,病害的发生对玉米造成极大为害,病毒病对玉米稳产高产已构成严重威胁。近年来,安徽、山东和辽宁玉米主要种植区病毒病危害较重。为了检测发病玉米的病毒种类,本研究利用小RNA高通量测序技术鉴定玉米病毒,明确种类,以期为制定抗病毒策略提供理论依据。据不完全统计,世界上有40多种玉米病毒病(http://en.wikipedia.org),在我国发生并报道的有5种,分别为玉米粗缩病、玉米矮花叶病、玉米条纹矮缩病、玉米红叶病和玉     

Extreme Reduction of Disease in Oats Transformed with the 5' Half of the Barley Yellow Dwarf Virus-PAV Genome

ABSTRACT Barley yellow dwarf viruses (BYDVs) are the most serious and widespread viruses of oats, barley, and wheat worldwide. Natural resistance is inadequate. Toward overcoming this limitation, we engineered virus-derived transgenic resistance in oat. Oat plants were transformed with the 5' half of the BYDV strain PAV genome, which includes the RNA-dependent RNA polymerase gene. In experiments on T2- and T3-generation plants descended from the same transformation event, all BYDV-inoculated plants containing the transgene showed disease symptoms initially, but recovered, flowered, and produced seed. In contrast, all but one of the BYDV-PAV-inoculated nontransgenic segregants died before reaching 25 cm in height. Although all of the recovered transgenic plants looked similar, the amount of virus and viral RNA ranged from substantial to undetectable levels. Thus, the transgene may act either by restricting virus accumulation or by a novel transgenic tolerance phenomenon. This work demonstrates a strategy for genetically stable transgenic resistance to BYDVs that should apply to all hosts of the virus.     

Yellow mottle of tomatillo (Physalis ixocarpa) caused by physalis mottle virus

Tomatillo ( Physalis ixocarpa ) is being evaluated as a food crop in Louisiana. Preliminary studies indicate that virus diseases may be the major factor limiting production. A foliar mosaic and yellow mottle disease was found commonly affecting plants in experimental plots. The cause of the disease was identified as physalis mottle virus (PhyMV). The virus was identified by host reaction, serology and dsRNA analysis. Other viruses found less frequently included cucumber mosaic virus and tomato spotted wilt virus. The flea beetle, Epitrix cucumeris , transmitted PhyMV experimentally. The annual weed Physalis pubescens was found naturally infected with PhyMV near infected tomatillo.     

RNAi‐based enhanced resistance to Cowpea severe mosaic virus and Cowpea aphid‐borne mosaic virus in transgenic cowpea

Cowpea (Vigna unguiculata) is one of the most important legumes cultivated in many parts of the world. The diseases caused by Cowpea severe mosaic virus (CPSMV) and Cowpea aphid‐borne mosaic virus (CABMV) are considered among the most important constraints on yield and quality, especially in Latin America and Africa. Here, the concept of using an RNA interference construct to silence the CPSMV proteinase cofactor gene and the CABMV coat protein gene is explored, in order to generate resistant transgenic cowpea plants. Ten cowpea transgenic lines were produced, presenting a normal phenotype and transferring the transgene to the next generation. Plants were tested for resistance to both CABMV and CPSMV by mechanical co‐inoculation. Seven lines presented milder symptoms when compared to the control and three lines presented enhanced resistance to both viruses. Northern analyses were carried out to detect the transgene‐derived small interfering RNA (siRNA) in leaves and revealed no correlation between siRNA levels and virus resistance. Additionally, in the symptomless resistant lines the resistance was homozygosis‐dependent. Only homozygous plants remained uninfected while hemizygous plants presented milder symptoms.     

甘蔗病毒病及其防治研究进展

甘蔗是最重要的糖料作物,由于其栽培过程中采用种茎无性繁殖,病毒病发生逐年加重.已知侵染甘蔗的病毒种类有甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)、高粱花叶病毒(Sorghummosaic virus,SrMV)、甘蔗线条花叶病毒(Sugarcane streakmosaic virus,SCSMV)、甘蔗黄叶病毒(Sugarcane yellow leaf virus,SCYLV)、甘蔗斐济病病毒(Sugarcane Fiji disease virus,SFDV)、甘蔗线.条病毒(Sugarcane streak virus,SSV)和甘蔗杆状病毒(Sugarcane bacilliform virus,SCBV).文中简要介绍上述几种病毒的基本特性及其所致病害的发生特点,对目前甘蔗病毒病防治技术进行了评述,提出了我国甘蔗病毒研究中需要关注的若干问题.     

表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染

 利用RT-PCR分别克隆了CMV P3613株系的RNA2片段、MP(movement protein)基因片段及CMV AN株系的CP(coat protein)基因片段。以CP基因为中间间隔序列,分别构建了含有RNA2片段和MP基因反向重复片段的原核表达载体。体外转录试验表明:两个载体转录后都能形成预期大小的dsRNA。经过IPTG诱导,在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase和RNaseA消化处理,证实为dsRNA。将表达病毒基因dsRNA的细菌超声破碎后处理烟草,进行保护和治疗试验,结果表明:表达CMV MP基因和RNA2片段dsRNA的细菌破碎液能够诱导烟草对CMV产生抗性。接种病毒60d后,保护效果试验病株率分别为45%和60%,治疗效果试验病株率分别为75%和85%,而其他对照发病率均为100%。本研究结果证明了利用RNA沉默的原理,构建具有反向重复序列的原核表达载体,用细菌表达dsRNA的粗提取物可防治CMV对烟草的侵染。     

Biology of a new virus isolated from Lupinus nootkatensis plants in Alaska

A new virus named Nootka lupine vein-clearing virus (NLVCV) was isolated from Lupinus nootkatensis plants that were confined to a relatively small area in the Talkeetna mountains of south-central Alaska. Annual surveys (2000–03) consistently found leaf symptoms of pronounced vein clearing and mosaic on 3- to 4-week-old plants in late June. Spherical particles ≈30 nm in diameter were isolated from these leaves. Virions contained a single-stranded RNA of ≈4·0–4·2 kb and one species of capsid protein estimated to be ≈40 kDa. The double-stranded RNA profile from naturally infected leaves consisted of three major bands ≈4·2, 1·9 and 1·5 kbp. Protein extractions from either sap or virions of diseased plants reacted to polyclonal antiserum made against the virions in Western blot assays. A predicted PCR product ≈500 bp was synthesized from virion RNA using primers specific to the carmovirus RNA-dependent RNA polymerase (RDRP) gene. The nucleotide sequence of the amplified DNA did not match any known virus, but contained short regions of identity to several carmoviruses. Only species belonging to the Fabaceae were susceptible to NLVCV by mechanical inoculation. Based on dsRNA profile, size of virion RNA genome and capsid protein, and similarity of the RDRP gene to that of other carmoviruses, it is suggested that NLVCV is a member of the family Tombusviridae , and tentatively of the genus Carmovirus . As the host range, RDRP gene and dsRNA profile of NLVCV are different from those of known viruses, this is a newly described plant virus.     

正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究

 RNA介导的病毒抗性与RNA沉默现象密切相关。反向重复cDNA序列(IR)的转录产物往往形成双链RNA结构,而双链RNA是诱发RNA沉默的有效因子。据此,本研究通过体外合成马铃薯Y病毒坏死株系衣壳蛋白基因(PVY^N-CP)5'端反向重复cDNA序列和正向重复cDNA序列(DR),分别构建植物表达载体pROK-IR和pROK-DR,利用农杆菌介导方法转化烟草NC89,比较这2种转基因烟草在RNA介导抗病性方面的差异。抗病性检测表明,转化IR和DR的转基因烟草均可获得抗病程度达到免疫的植株,但转化IR序列可大大提高抗病植株在转基因植株中的比例。分析结果表明所获得的抗病性为RNA介导的抗病性,是RNA沉默的结果。这一研究结果为利用IR策略进行抗病毒遗传育种提供了理论依据,并为讲一步开展RNA介导抗病性的机制研究奠宗了基础。     

Avoiding Secondary Leaf Fall Disease of Rubber by Chemical Defoliation

Abstract

An intensive survey of cultivated plants throughout the island of Montserrat resulted in the identification of a number of viruses. Squash mosaic virus was found in cantaloupe, pumpkin and squash. There were cases of multiple infection of pumpkin and squash by cucumber and squash mosaic viruses. Potato virus Y was recovered from pepper and tomato. Bean yellow mosaic and cowpea mosaic viruses were found in red kidney bean and string bean while cowpea mosaic virus was found in cowpea and broad bean (Jamaica bean). Other viruses that were previously identified on the basis of field symptomatology were confirmed by host range and serology. Viruses were not recovered from sea island cotton or from sweet potato, although field symptoms on the sweet potato are similar to those caused by sweet potato mosaic virus. The leaf tissues of crop plants were inoculated to a wide range of indicator plants. Some of those that reacted systemically were examined by electron microscopy for virus particles and the sap extracted from them was used in serodiagnostic assays for virus identification. This is the first attempt to identify positively the viruses of crop plants on the island.     

Lichens—a new source or yet unknown host of herbaceous plant viruses?

Lichens are symbiotic associations of fungi with green algae or cyanobacteria. They have arisen independently several times within the Ascomycota and Basidiomycota. This symbiosis became with time one of the most successful life forms on Earth. Outside of the symbiotic algae and fungi, there are endophytic fungi, other algae, and lichen-associated bacteria present within lichen thalli. Till now, no lichen-specific pathogens have been reported among bacteria and viruses. Around 15 dsRNA viruses are known from Eurotiomycetes and another dsRNA and reverse transcribed ssRNA viruses from Dothideomycetes containing some lichenized fungal lineages. Algal viruses have been identified from less than 1 % of known eukaryotic algal species but no virus has been found in Trebouxia or in Trentepohlia (Chlorophyta, Pleurastrophyceae, Pleurastrales), the most common green lichen photobionts. On the other hand, dsDNA viruses infecting related Chlorella algae are well known from freshwater phytoplankton. However, high-molecular weight dsRNA isolated from different lichen thalli indicated to us presence of ss or dsRNA viruses. A PCR-based search for viruses with genus-specific and species-specific primers resulted in amplification of genome segments highly identical with those of plant cytorhabdoviruses and with Apple mosaic virus (ApMV). The nucleotide sequence of the putative lichen cytorhabdovirus showed high identity (98 %) with Ivy latent cytorhabdovirus. The nucleotide sequences of six Apple mosaic virus isolates from lichens showed high similarity with ApMV isolates from apple and pear hosts. The lichen ApMV isolates were mechanically transmitted to an herbaceous host and detected positive in ELISA 14 days thereafter, which support its infectivity on plants. We prepared axenic cultures of photobionts identified as Trebouxia sp. from this ApMV-positive lichen samples. All these cultures were positive for ApMV in RT-PCR test. We suggest that lichens as a whole (or their photobionts, more specifically) could serve as reservoirs for viruses, despite the fact that the way of transmission between different organisms is not clear. We showed that lichens could harbour several viruses simultaneously, as the plant cytorhabdovirus and ApMV were detected in the same host, also.     

The coat proteins and putative movement proteins of isolates of Prunus necrotic ringspot virus from different host species and geographic origins are extensively conserved

A complete sequence for the RNA 3 of Prunus necrotic ringspot virus (PNRSV) is described (Genbank Accession U57046). Primers from this sequence were used to amplify both the movement protein and coat protein genes of 3 other isolates of PNRSV originating from different host species and geographic locations. Comparisons of these sequences with those of other published sequences for PNRSV and the closely related apple mosaic virus (ApMV) showed that both the movement proteins and coat proteins of isolates of PNRSV are extensively conserved irrespective of either the original host or the geographic origin. The movement protein and coat protein of ApMV and PNRSV are sufficiently conserved to suggest that these two viruses may have evolved from a common ancestor. The amino acid sequence of the two coat proteins shows areas of similarity and difference that would explain the serological continuum reported to occur among isolates of these two viruses. Nevertheless, the movement protein and coat protein of the two viruses are sufficiently different so that ApMV and PNRSV should be considered to be distinct viruses.     

RNAi‐mediated plant protection against aphids

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double‐stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant‐mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant‐mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant‐mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid‐resistant plants through plant‐mediated RNAi strategy. © 2016 Society of Chemical Industry     

A short‐length single chimeric transgene induces simultaneous silencing of Agrobacterium tumefaciens oncogenes and resistance to crown gall

Transformation with self‐complementary oncogene sequences was used to silence the Agrobacterium tumefaciens oncogenes ipt and iaaM. The silencing response was triggered by using a very short chimeric sequence where conserved fragments from both oncogenes were fused in one unique transgene. Most T₀ transgenic tobacco lines and T₁ seedlings evaluated in vitro had intermediate or very low susceptibility to A. tumefaciens as compared with the wildtype plants. A greenhouse evaluation of whole plants confirmed the lines that were resistant. Low levels of transgene hairpin RNA (hpRNA) coupled with small interfering RNA (siRNA) accumulation correlated with oncogene silencing and, therefore, resistance to crown gall. After infection with the oncogenic strain, much lower levels of the oncogenes’ mRNA were found in resistant lines than in wildtype plants. The frequency of resistant lines, with few or no symptoms, produced with the chimeric construct was similar to the highest reported efficiencies obtained by using sense and antisense whole oncogene sequences.     

Production of anti-virus,viroid plants by genetic manipulations

Many pathogenic plant viruses are RNA viruses, which initiate production of double-stranded RNA intermediates when they replicate in host plant cells. Introduction of double-stranded RNA-specific ribonucleases such as the Schizosaccharomyces pombe derived pac I protein and animal cell derived interferon-induced 2',5'-oligoadenylate synthetase (2-5 Aase)/ribonuclease L (RNase L) system into various plants may make plants resistant to various pathogenic viruses and viroids. We have demonstrated that pac I and 2-5 Aase/RNase L transgenic tobacco plants are resistant to various viruses including tobacco mosaic virus, cucumber mosaic virus and potato virus Y. In addition, pac I transgenic potato plants are resistant to potato spindle tuber viroid. Using Agrobacterium-mediated transformation, we have established a transformation system for chrysanthemum plants and have recently developed pac I transgenic chrysanthemum (Dendranthema grandiflora cv Reagan) resistant to chrysanthemum stunt viroid and have grown them in isolated fields for an evaluation of their effects.     

侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用

 根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg²⁺浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。