The Helper Component-Proteinase of Sweet potato feathery mottle virus Facilitates Systemic Spread of Potato virus X in Ipomoea nil

ABSTRACT When Ipomoea nil was coinfected with Sweet potato feathery mottle virus (SPFMV), a member of the genus Potyvirus, and Potato virus X (PVX) typical symptoms caused by PVX were observed on those by SPFMV on the first upper true leaves at 14 days postinoculation (dpi). On the other hand, no PVX-induced symptoms were observed on the first upper true leaves at 14 dpi when plants were infected with PVX alone. In the case of coinfection with PVX and SPFMV, PVX RNA was detected not only in the inoculated cotyledonary leaves but also in the first upper true leaves at 14 dpi. In the case of single infection with PVX, PVX RNA was detected in the inoculated cotyledonary leaves but not in the first upper true leaves at 14 dpi. The accumulation of SPFMV remained unchanged, regardless of whether the inoculum consisted of SPFMV alone or a mixture of SPFMV and PVX. Although recombinant PVX engineered to express the helper component-proteinase (HC-Pro) of SPFMV (PVX.HC) enhanced symptoms severity in Nicotiana benthamiana, PVX.HC induced the synergism characterized by an enhanced viral movement in Ipomoea nil. Immunofluorescence microscopic examination revealed that the HC-Pro was present in phloem of SPFMV-infected I. nil. These results suggest that SPFMV HC-Pro acts as an enhancer of long distance movement for PVX in I. nil.     

黄瓜绿斑驳花叶病毒低风险参照物质的研制

采用RT-PCR方法扩增黄瓜绿斑驳花叶病毒的外壳蛋白基因与3,非编码区,并将其构建到马铃薯X病毒(PVX)载体中.重组质粒经线性化及体外转录后接种烟草,获得含有病毒外壳蛋白基因的感病植株.感病组织可用于分子检测的质控,也可作为毒源来繁殖阳性参照物质.基于PVX载体制备的参照物质可降低检疫性病毒的生物安全风险,尤其对稳定性强、可造成严重经济损失的高风险病毒的检疫更具实用价值.     

Diverse conserved domains and a positively selected site in the sugarcane streak mosaic virus P1 protein are essential for RNA silencing suppression and protein stability

RNA silencing is one of the conserved antiviral mechanisms in plants, and viruses encode RNA silencing suppressors (RSS) to overcome host RNA silencing and facilitate virus infection. Sugarcane streak mosaic virus (SCSMV; species Sugarcane streak mosaic virus, genus Poacevirus, family Potyviridae) is a major causal agent of sugarcane mosaic disease in many countries in Asia, including China. In this study, we used Agrobacterium co-infiltration to show that the SCSMV P1 protein, rather than the helper component-proteinase (HC-Pro), functions as a strong RSS to suppress local RNA silencing in Nicotiana benthamiana. Mutational analysis indicated that the 15 amino acids (aa; aa 1–15) of the SCSMV P1 N-terminus were not important for RNA silencing suppression, but rather another 15 aa domain (aa 108–122) containing a conserved motif (LFR/KNKQAYIST) was essential for efficient silencing suppression by P1. In addition to the 15 aa (aa 344–358) domain in the P1 N-terminus, another 15 aa domain (aa 65–79) of P1, containing the LXKA motif and one conserved aa (D78), were associated with P1 protein stability. Furthermore, substituting the histidine (H263) residue in P1 with threonine (H263T) or alanine (H263A) also affected P1 protein stability. Notably, the H263 residue is both a positively selected site and part of the serine protease catalytic triad (HDS). Taken together, our data demonstrate that SCSMV P1, and not HC-Pro, plays a functional role in suppressing RNA silencing, and also show that some conserved motifs and a positivelyselected site in the P1 protein are associated with RSS activity and protein stability.     

HC Pro 3个参与抑制RNA沉默氨基酸位点的鉴定

 马铃薯Y病毒属病毒编码的辅助成分-蛋白酶（helper component-proteinase,HC-Pro）是第一个被鉴定的RNA沉默抑制因子。本研究通过定点突变的方法获得了马铃薯A病毒（Potato virus A,PVA）HC-Pro的3个突变体, 利用农杆菌共浸润的方法分析了这些突变对HC-Pro抑制RNA沉默活性的影响。与野生型HC-Pro处理相比,Phe⁶、Asn¹¹ 缺失突变体的处理中绿色荧光减弱,而Ile²⁵⁰-Gly²⁵¹-Asn²⁵²（IGN）基序中的Ile和Gly分别突变为Asp和Glu的处理中观察不到绿色荧光。该结果表明Phe⁶、Asn¹¹ 和IGN²⁵⁰  3个位点均参与调控HC-Pro的抑制RNA沉默活性。     

马铃薯X病毒荧光定量PCR检测体系的建立及应用

马铃薯X病毒(Potato virus X,PVX)是危害茄科作物的一种重要病毒,为了建立特异性检测PVX的实时荧光定量PCR体系,本研究以PVX-1985分离物中外壳蛋白(coat protein,CP)基因序列为模板,设计引物构建重组质粒并选择扩增效率高、特异性强的引物成功构建出标准曲线。利用建立的体系,成功检测到以含pCaPVX440侵染性克隆载体的农杆菌C58C1接种后的本氏烟(Nicotiana benthamiana)中PVX病毒RNA的拷贝数。     

高温激活RNA沉默介导的心叶烟抗病毒防御反应

 在真核细胞内,RNA沉默是一种保守的抵抗病毒及外源基因等寄生分子的防御机制。温度可以明显地影响植物体与病毒之间的相互作用。在常温状态下,常常会伴随病毒病的爆发,但在较高温度下,症状则会减轻,甚至有些感病植株新生叶片可以较快地恢复健康。目前这类现象的内在机制还不完全清楚。本文通过定量分析不同温度下马铃薯X病毒(Potato virus X,PVX)和马铃薯Y病毒(Potato virus Y,PVY)侵染的心叶烟(Nicotiana glutinosa)植株体内病毒蛋白含量,结合症状观察,确定30℃左右是PVX和PVY在心叶烟上引起症状消失即发生系统性RNA沉默的临界温度,此时的感病植株上部新生展开叶片呈现无症状态,下部已发病叶片症状不变。在常温时,由病毒激发的RNA沉默会受到抑制,病毒来源的小分子干涉RNA (small interfering RNAs,siRNAs)含量极少或根本检测不到,植株对于病毒的敏感性较强。当温度达到30或32℃时,感病植株下部症状保持叶片中,病毒起源的siRNAs含量明显增加,而上部新生健康叶片中却检测不到siRNAs的存在。这说明高温激活了RNA沉默介导的植物抗病毒防御反应,导致病毒症状减轻甚至消失。     

Hosts and symptoms of Plum pox virus: Herbaceous hosts

Plum pox virus (PPV) is polyphagous and epidemic. Apart from cultivated and wild Prunus species, a large number of herbaceous plants can be hosts of the virus. New herbaceous host species are continuously being reported following artificial inoculation studies. Some of these herbaceous hosts, Chenopodium foetidum , Nicotiana clevelandii , N. benthamiana and Pisum sativum are very useful for concentrating and purifying the virus. The list of plants that have been found to be infected with PPV in their natural environment is shorter than the list of plants which can be experimentally infected. The role of weed species in PPV survival and spread in orchards is poorly understood. It is widely accepted that annual plants or weeds are not important in the epidemiology of PPV.     

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

Attenuated viruses have been isolated and studied not only as a practical means of controlling virus diseases but also to gain a molecular understanding of viral virulence and cross protection. They have been isolated from crop fields and generated through high/low temperature treatment or by mutagens such as nitrous acid and ultraviolet irradiation. Some viruses have been beneficially used in fields and evaluated for one or more decades. Molecular genetic studies on attenuated viruses have revealed that amino acid substitutions are located in replicase and the movement protein in tobamovirus, protein 2b for cucumovirus, and P1 and HC-Pro for potyvirus. In most cases, with a few exceptions, symptom attenuation is positively correlated with a reduced level of RNA silencing suppression. Molecular mechanisms underlying virus attenuation and cross protection and the rationale for practical use of attenuated viruses for effective virus disease control are discussed.     

Soybean mosaic virus Helper Component-Protease Alters Leaf Morphology and Reduces Seed Production in Transgenic Soybean Plants

ABSTRACT Transgenic soybean (Glycine max) plants expressing Soybean mosaic virus (SMV) helper component-protease (HC-Pro) showed altered vegetative and reproductive phenotypes and responses to SMV infection. When inoculated with SMV, transgenic plants expressing the lowest level of HC-Pro mRNA and those transformed with the vector alone initially showed mild SMV symptoms. Plants that accumulated the highest level of SMV HC-Pro mRNA showed very severe SMV symptoms initially, but after 2 weeks symptoms disappeared, and SMV titers were greatly reduced. Analysis of SMV RNA abundance over time with region-specific probes showed that the HC-Pro region of the SMV genome was degraded before the coat protein region. Transgenic soybean plants that expressed SMV HC-Pro showed dose-dependent alterations in unifoliate leaf morphologies and seed production where plants expressing the highest levels of HC-Pro had the most deformed leaves and the lowest seed production. Accumulation of microRNAs (miRNAs) and mRNAs putatively targeted by miRNAs was analyzed in leaves and flowers of healthy, HC-Pro-transgenic, and SMV-infected plants. Neither expression of SMV HC-Pro nor SMV infection produced greater than twofold changes in accumulation of six miRNAs. In contrast, SMV infection was associated with twofold or greater increases in the accumulation of four of seven miRNA-targeted mRNAs tested.     

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     

马铃薯Y病毒HC-Pro第182位赖氨酸残基参与引起烟草叶脉坏死

马铃薯Y病毒(potato virus Y,PVY)是侵染烟草的最重要病毒之一.不同PVY株系侵染烟草可引起不同症状,有些PVY株系可引起烟草叶脉坏死,严重影响烟草的产量和品质.PVY A12分离物属于NTN-NW株系,但侵染珊西烟(Nicoti-ana tabacum cv.Xanthi)不能引起叶脉坏死.分析发现,...     

RNA沉默转基因技术在植物抗病虫害研究中的作用

RNA沉默技术由于其简便、高效及高特异性在各种模式和非模式生物系统中得到了广泛的应用。植物转基因技术是将植物、动物或微生物中分离到的目的基因,通过各种手段整合到植物基因组上,使其稳定遗传并赋予植物新的优良性状的生物技术。RNA沉默在植物转基因工程领域的研究十分广泛,目前已经成功研究培育出许多抗病毒、抗虫转基因植物,为农业病虫害的防控提供了新的思路和方法。本文重点综述了近年来植物转基因介导RNA沉默在抗虫与抗病毒研究方面的进展,加深人们对转基因植物抗虫与抗病毒研究的认识。     

Significance of the heterologous encapsidation of zucchini yellow mosaic potyvirus in transgenic plants expressing plum pox potyvirus capsid protein1

Transgenic Nicotiana benthamiana plants expressing the coat protein of an aphid-transmissible strain of plum pox potyvirus (PPV-D) were infected with an aphid non-transmissible strain of another potyvirus, zucchini yellow mosaic potyvirus (ZYMV-NAT). Non-viruliferous Myzus persicae could acquire and transmit ZYMV-NAT from these plants but not from infected N. benthamiana control plants (not transformed, or transformed by the vector alone). Immunosorbent electron microscopy experiments using the decoration technique revealed that ZYMV-NAT virus particles in the infected transgenic plants expressing the PPV coat protein could be coated not only with ZYMV antibodies but also, on segments of the particles, with PPV antibodies. This suggests that aphid transmission of ZYMV-NAT occurred through heterologous encapsidation, and reveals a potential risk of releasing genetically engineered plants expressing viral coat proteins into the environment.     

Mature plant resistance of potato against some virus diseases. I. Concurrence of development of mature plant resistance against potato virus X,and decrease of ribosome and RNA content

Four of five weeks after planting a group of potato plants ‘Bintje’ was inoculated with potato virus X (PVX). Other groups were inoculated at intervals of 14 days. Tubers produced by plants inoculated 35 days after planting were all infected. The plants inoculated 49 days or later after planting produced few infected tubers. The latter had developed mature plant resistance against PVX infection. The ribosome and RNA contents of leaves were measured by application of adsorption chromatography. A rapid decrease in ribosome and RNA contents occurred in plants at the time of rapid increase in the rate of mature plant resistance. The decrease was most distinct in the fifteenth leaf and therefore the contents in this leaf seem to give a good indication of the rapid increase in resistance.