Amino acid 129 in the coat protein of Cucumber mosaic virus primarily determines invasion of the shoot apical meristem of tobacco plants

We previously reported that a strain of Cucumber mosaic virus (Pepo CMV) invaded the shoot apical meristem (SAM, tunica corpus) of tobacco plants at 6–8 days postinoculation (dpi), contrary to earlier observations. To identify a viral factor determining the ability to invade the SAM, we inoculated plants with two other CMV strains, MY17 and Y, and tested the three strains in this study. Immunohistochemical microscopy revealed that MY17 CMV invaded the SAM at 7 dpi, the same as Pepo CMV, but Y CMV did not, even at 21 dpi. Using RNA pseudorecombinants between Pepo and Y CMV, we found that Pepo RNA 2 affected the rate of SAM invasion, and Pepo RNA 3 was required for successful SAM invasion. Inoculation with RNA 1 and RNA 2 from Y CMV and RNA 3 containing the chimeric coat protein (CP) gene between Pepo and Y CMV or a Y RNA 3 point mutant containing a Ser-to-Pro substitution at position 129 in CP (Y129P) revealed that amino acid 129 of CP is the determinant for successful SAM invasion. The rate of SAM invasion of the pseudorecombinants and Y129P was consistent with the efficiency of cell-to-cell movement in the inoculated leaves, implying that SAM invasion by CMV strains may be due to efficient cell-to-cell movement.     

黄瓜花叶病毒M株系引致烟草症状恢复的初步研究

 黄瓜花叶病毒M株系在白肋烟上具有典型的症状恢复现象,本研究用提纯病毒和DAS-ELISA建立了CMV-M病毒定量检测方法。研究发现,症状严重程度与叶片中的病毒浓度呈正相关:最早发病的黄化叶每克病组织中病毒可高达790 μg,而恢复叶片上部再发病的花叶症状病叶中每克病组织中病毒也可高达508 μg,恢复叶片中病毒含量很低,每克叶片中最高也没有超过6 μg,仅为发病叶片病毒浓度的1/85~1/135,远低于根和茎中的病毒浓度。RT-PCR和生物学检测结果表明恢复叶片中确实存在具侵染活性的病毒,而且病毒在长达半个月以上一直保持很低浓度。结果表明恢复叶中可能存在有效的病毒防御机制,其具体机理有待进一步研究。     

2b Protein is essential to induce a novel gradual cell death in Zucchini yellow mosaic virus-inoculated cucumber cotyledon co-infected with Cucumber mosaic virus

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

Cucumber mosaic virus isolated from Aconitum spp. in Japan

Viruses were isolated from leaves of plants of Aconitum species with symptoms such as mottling and yellowing in Hokkaido and Gunma prefectures in Japan. These viruses were identified as Cucumber mosaic virus (subgroup II) based on particle morphology, host range, aphid transmission, and serology.     

Analysis of natural populations and possible natural reassortment of Cucumber mosaic virus

Plants naturally infected with Cucumber mosaic virus (CMV) were collected and analyzed by electrophoresis of the replicative form of dsRNA and by Northern blot hybridization using CMV RNA-specific probes. Some of the CMV-infected plants, especially winter crops, contained two kinds of RNA 1 segments or RNA 2 segments (or both), suggesting that mixed infections of CMV occurred naturally. Single-aphid-transmitted isolates (SATIs) from the field isolate containing two RNA 1 segments were grouped into three types by the electrophoretic mobility of RNA 1 (i.e., those containing one slow segment, those containing one fast segment, and those containing both). Furthermore, SATIs and single-lesion isolates, generated from the plants inoculated with a mixture of two CMV isolates that could be differentiated by their electrophoretic dsRNA profiles, were analyzed by dsRNA, indicating that nonparental progenies were observed. These results suggested that genetic reassortment of CMV RNA may occur in nature and that this is an important mechanism in CMV evolution.     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

超敏蛋白对烟草黄瓜花叶病毒的诱导抗性及生长、品质的影响

为明确超敏蛋白对烟草黄瓜花叶病毒(Cucumber mosaic virus,CMV)的诱导抗性及其生长、品质的影响,采用半叶枯斑法和田间试验研究了10%超敏蛋白对枯斑三生烟和普通烟的诱导抗性及促生作用,并分析了对烤烟经济性状和内在质量的影响。结果表明,10%超敏蛋白在活体外对CMV无钝化作用,但预防、治疗效果显著,接种前48 h喷施预防效果达75.70%,接种后24 h喷施治疗效果达65.45%,均显著高于对照;10%超敏蛋白处理后烟草的株高、茎围、最大叶面积均高于其它处理,单位产量比20%吗啉胍·乙铜可湿性粉剂处理增产211.8 kg/hm2,增值3 519.2元/hm2;经10%超敏蛋白处理后的烤烟化学成分均处于优质烟的适宜质量分数范围内。表明超敏蛋白不仅有利于提高烟草抗性和促进烟株生长,且可以明显提高烟叶经济效益和烟叶内在质量。     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

黄瓜花叶病毒和马铃薯Y病毒混合侵染烟株对烟蚜取食行为的影响

为探究黄瓜花叶病毒(Cucumber mosaic virus,CMV)、马铃薯Y病毒(Potato virus Y,PVY)混合侵染烟株对烟蚜取食行为的影响,利用刺探电位图谱(electrical penetration graph,EPG)技术记录了烟蚜在健康烟株与CMV、PVY混合侵染后不同发病级别烟株上的取食波形。结果显示:烟蚜在健康烟株上的刺探次数最少,在感病烟株上的C波总持续时间均显著长于健康烟株;第1次到达韧皮部前的刺探次数,健康植株上仅为4.00次,3级感病烟株上的为健康烟株上的2倍;在健康烟株上E2波总持续时间为120.65 min,极显著大于2级和3级感病烟株;刺探过程中,感病烟株上的pd波出现次数均高于健康烟株,且pd波II-1和II-3亚波的持续时间也显著高于健康烟株。研究表明,CMV、PVY混合侵染烟株可降低寄主对烟蚜的适合度,且能促进烟蚜对病毒的传播。     

白色霞水母刺细胞毒素抗烟草花叶病毒活性

为探索从水母毒素中获取抗植物病毒物质的应用前景,采用半叶枯斑法、整株法和漂浮叶圆片法,对白色霞水母Cyanea nozakii Kishinouye刺细胞毒素的抗烟草花叶病毒(Tobacco mosaic virus,TMV)活性进行了检测。结果表明,白色霞水母刺细胞毒素对TMV具有很强的直接钝化作用,枯斑抑制率随钝化时间的延长而增大。0.99 mg/m L毒素体外钝化TMV 30 min,枯斑抑制率达98.85%,将TMV与0.80 mg/m L毒素体外混合后立即接种,枯斑抑制率仍达89.57%;20~70℃之间,毒素对TMV的钝化作用随温度升高而降低,但又表现出一定的高温耐受性,70℃下处理30min,1.33 mg/m L毒素对枯斑的抑制率为61.73%。毒素对病毒初侵染有一定的预防作用,并可抑制TMV的增殖;1.93 mg/m L毒素处理接种TMV的叶圆片2 d后,对TMV的增殖抑制率为49.41%;但该毒素对TMV所致病害的治疗效果不明显。该毒素还具有较强的蛋白水解酶活性,可能与其抗TMV活性相关。表明白色霞水母刺细胞毒素抗TMV作用明显,其抗植物病毒活性值得深入研究。     

Characterization and detection ofCucumber green mottle mosaic virus in Greece

The host range specificity of Greek isolates ofCucumber green mottle mosaic virus (CGMMV) was determined and the ability of the virus to retain its infectivity in naturally contaminated soil, after storage at 4°C for at least 10 months, was established. An immunocapture RT-PCR protocol was developed for the detection of the virus and proved to be 10⁵ times more sensitive than DAS-ELISA and 10² times more sensitive than F(ab’)₂-ELISA using F(ab’)₂ fragments that were prepared from an antiserum, which was raised against a local virus isolate. In order to clarify dissimilarities that were revelaed between the coat protein (CP) genes of the Greek isolates after restriction mapping of their respective PCR products, the CP genes of three isolates were sequenced and found to be very similar but not identical to the CP gene of CGMMV-SH. http://www.phytoparasitica.org posting Aug. 27, 2002.     

人工接种CGMMV的增殖及其对西瓜 光合作用的影响

为明确西瓜感染黄瓜绿斑驳花叶病毒CGMMV后病毒的增殖过程及其对光合作用的影响,本研究以辽宁省田间主栽西瓜品种‘京欣’为试材,测定了人工接种CGMMV后,西瓜植株体内病毒含量、光合色素、光合作用相关指标以及参与光合作用的关键酶活性的变化,结果显示:接种3d后,即可检测到病毒,24d植株内病毒含量达最高;叶绿素a、叶绿素b及类胡萝卜素的含量均有所降低,叶绿素a在9d后均低于健康对照,而叶绿素b和类胡萝卜素在15d后明显低于健康对照,第18天时,健康对照的叶绿素b含量达接种处理的1.33倍;ALA脱水酶活性除第15天与对照无显著差异外,整个测定期间均低于健康对照,21d时健康对照的ALA脱水酶的活性达29.9U/h,为接种处理的2.18倍;PEP羧化酶的活性显著低于健康对照,测定期间接种处理的PEP羧化酶活性变化不大;乙醇酸氧化酶活性明显升高,测定期间其活性均显著高于健康对照;西瓜叶片的光合速率、气孔导度和蒸腾速率均远小于健康对照,而细胞间隙CO_2浓度却比对照高,本研究为揭示西瓜感染CGMMV后的光合作用变化规律奠定了基础。     

两种类型黄瓜花叶病毒卫星RNA的序列、结构以及对辅助病毒的致病性调控分析

黄瓜花叶病毒(Cucumber mosaic virus,CMV)卫星RNA(satellite RNA,satRNA)通常影响CMV的致病性。本研究设计了检测satCMV的简并引物,通过RT-PCR从山东省泰安市3种自然发病寄主中检测到satCMV,发现了两类长度有明显差异的satCMV:白菜与萝卜中克隆的satCMV为339个核苷酸,二者的核苷酸一致率为99.41%;烟草中克隆的satCMV为383个核苷酸,与白菜和萝卜中satCMV的核苷酸序列一致率均为71.06%。系统进化分析表明这两类长度有明显差异的satCMV亲缘关系较远。两种类型的satCMV与CMV-Fny混合接种对CMV致病症状的调控存在明显差异。利用m Fold软件分析了两类不同长度的CMV卫星RNA基因组及其互补链的RNA结构特征,表明两类sat CMV与CMV互作的活跃度以及核心结构域存在差异。两类satCMV全基因组的克隆以及结构分析为研究不同类型satCMV与CMV的互作及其对CMV致病性的影响奠定了基础。     

Accumulation of short interfering RNAs characteristic of RNA silencing precedes recovery of tomato plants from severe symptoms of <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> infection

Tomato plants infected with Potato spindle tuber viroid (PSTVd) had severe leaf curling and vein necrosis. The disease symptoms began to diminish during the late stages of infection, however, and almost healthy-looking leaves began to appear on the upper portion of the plants. PSTVd concentrations reached their highest levels in leaves with severe symptoms and decreased in upper leaves recovering from severe symptoms. PSTVd-specific short interfering RNAs (siRNAs), characteristic of RNA silencing, accumulated in all leaves in which PSTVd reached a detectable level, suggesting that recovery from severe disease was induced by RNA silencing via sequence-specific degradation of PSTVd.     

西瓜感染黄瓜绿斑驳花叶病毒后糖分代谢与倒瓤的关系

为揭示人工接种黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)后西瓜糖分代谢与倒瓤的关系,采用高效液相色谱法测定了接种CGMMV后西瓜的葡萄糖、果糖、蔗糖含量变化,采用比色法测定了糖酵解途径关键酶及蔗糖代谢相关酶活性的变化。结果显示,西瓜苗感染CGMMV后,果实中葡萄糖、果糖、蔗糖含量均较对照发生明显变化,接种处理后葡萄糖含量在授粉7~28 d显著高于对照,之后迅速下降,35 d时显著低于对照,仅为对照的24.87%;果糖含量仅在授粉后7~14 d高于对照,21~35 d显著低于对照;接种处理和对照的蔗糖含量在授粉后14~35 d均逐渐上升,但14~35 d接种处理显著低于对照。接种处理后己糖激酶、磷酸果糖激酶和果糖激酶活性均在授粉后7~14 d显著低于对照,21~28 d显著高于对照,35 d时显著低于对照;丙酮酸激酶活性授粉后7~21 d显著低于对照,35 d时显著高于对照;酸性转化酶和中性转化酶活性在授粉后7~21 d显著低于对照,35 d时高于对照;蔗糖磷酸合成酶和蔗糖合成酶活性均显著低于对照,其中蔗糖合成酶活性在授粉后28 d时仅为对照的21.32%。推断西瓜苗接种CGMMV后糖酵解的4个关键酶协同作用,调控糖酵解,进而调控葡萄糖和果糖的积累与代谢,同时蔗糖代谢相关酶活性降低,阻碍蔗糖合成,即西瓜苗感染CGMMV导致倒瓤与其糖分的合成与代谢密切相关。     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.