Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

RNA-dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

RNA-dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd-derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus-induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA-mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.     

带卫星RNA黄瓜花叶病毒保护接种的植物中病毒积累与基因组RNA合成及病状表达

本文报道了三生烟在接种带卫星RNA的黄瓜花叶病毒(CMV-S52)后,或接种后用强毒CMV攻击,其病状表现、组织中病毒含量,病毒基因组RNA合成与卫星RNA合成的关系.植物在接种CMV-S52后7～10天,所有接种植物都表现出不同程度的轻度花叶症状,此期正是组织内病毒含量最高,而卫星dsRNA占总dsRNA百分比较低时期.接种后不同时间的测定证明,组织内病毒基因组dsRNAl和dsRNA2的合成水平均较低,而卫星dsRNA则始终保持较高的合成水平;到接种后15天病状消失时,卫星dsRNA占总dsRNA合成百分比的84.79％之多. 用CMV-S52保护接种后再用强病毒攻击,植物中的病毒含量、病毒ssRNA合成不增加或稍有增加;而基因组dsRNA和卫星dsRNA合成都有增加,但卫星dsRNA合成仍占绝对优势.保护作用的效果,取决于攻强病毒时已存在于组织内的卫星RNA与基因组RNA合成量的相对比例。讨论了卫星RNA的保护机理。     

Transformed tomato plants express a satellite RNA of cucumber mosaic virus and produce lethal necrosis upon infection with viral RNA.

Tomato plants transformed with a single copy of a tomato necrosis causing satellite RNA of cucumber mosaic virus (CMV) express the satellite sequence, but the plants show no disease symptoms and have a normal appearance. Upon challenge infection of the F1 progeny with a CMV strain free of any detectable encapsidated satellite the plants accumulated single and double-stranded forms of satellite RNA and developed lethal necrosis.     

Differential effects of Cucumber mosaic virus satellite RNAs in the perturbation of microRNA-regulated gene expression in tomato

Viral infections generally cause disease symptoms by interfering with the microRNA (miRNA)-mediated regulation of gene expression of host plants. In tomato leaves, the accumulation levels of eleven miRNAs and ten target mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus (TAV)-Bj infections. The ability of CMV-Fny to interfere with miRNA pathway was dramatically suppressed in the addition of the benign satellite (sat) RNA variant (satYn12), but was slightly affected when CMV-Fny was co-inoculated with the aggressive satRNA variant (satT1). In plants harboring the infection of CMV-FnyΔ2b (a CMV-Fny 2b-deletion mutant), the unaltered miRNAs and target mRNAs levels compared with mock inoculated plants indicated that 2b ORF was essential for perturbation of miRNA metabolism. When the amounts of viral open reading frames (ORFs) in these infections were quantified, we found satYn12 caused a higher reduction of CMV-Fny accumulation levels than satT1. These results indicate the complex mechanism by which satRNAs participate in CMV-tomato interaction, and suggest that the severity of disease symptoms positively correlates to some extent with the perturbation of miRNA pathway in tomato.     

Grafting to manage infections of top stunting and necrogenic strains of cucumber mosaic virus in tomato

Cucumber mosaic virus (CMV) lists among the most important etiological agents of tomato diseases. Some isolates of CMV function as helper virus for replication, encapsidation and transmission of satellite RNAs (satRNA), which may exacerbate symptoms induced by CMV in certain hosts. Outbreaks of CMV strains supporting hypervirulent variants of satRNAs are recurrent in tomato with devastating effects on crop production and efficient control measures are still unavailable. In this study, we examined the dynamics of infection of the CMV strains tomato top stunting (TTS) and 77 supporting replication of satRNA variants that codetermine top stunting (TTS‐satRNA) and necrotic (77‐satRNA) phenotypes in two tomato cultivars denoted Solanum lycopersicum Manduria (Sl‐Ma) and S. lycopersicum UC82 (Sl‐UC). Sl‐Ma but not Sl‐UC recovered from disease symptoms induced by CMV‐TTS while both the cultivars succumbed to the infection of CMV‐77 and its necrogenic satRNA. Ability to recover of the Sl‐Ma plants was transmitted by grafting to the susceptible genotype Sl‐UC. More interestingly, recovery was observed also against the challenge inoculation of CMV plus 77‐satRNA in plants grafted on Sl‐Ma and in self‐grafted plants of both the Sl‐Ma and Sl‐UC cultivars. Analysis of small RNAs and genes of the defence plant response based on RNA interference (RNAi) suggested that RNAi is involved in the recovery of Sl‐Ma against CMV with hypervirulent satRNAs and in scions grafted on this rootstock. The response of Sl‐Ma to the inoculation of CMV‐77 plus 77‐satRNA was compared with that of the transgenic tomato line S. lycopersicum transgenic line UCTC5.9.2 that expresses constitutively the benign variant of the satRNA denoted Tfn‐satRNA. Comparative analysis suggested that the response may operate via similar mechanisms, which involve RNAi, the graft and the presence of the satRNA.     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.     

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid — host cell interaction

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - CEVd citrus exocortis viroid - CSVd chrysanthemum stunt viroid - PSTVd potato spindle tuber viroid - TMV tobacco mosaic virus - phc photoheterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture     

A variant of the satellite RNA of groundnut rosette virus that induces brilliant yellow blotch mosaic symptoms in Nicotiana benthamiana

Some Malawian cultures of groundnut rosette virus (GRV) give rise to variants that, although still causing symptoms of the chlorotic type of rosette in groundnut, induce brilliant yellow blotch mosaic symptoms, instead of the usual veinal chlorosis and mild mottle, in Nicotiana benthamiana. One such isolate (YB) induced the formation in infected plants of a 0.9 kbp dsRNA having extensive sequence homology with molecules of similar size in other naturally occurring isolates of GRV. These dsRNA molecules were shown to be double-stranded forms of single-stranded satellite RNA molecules. Experiments in which the satellite was removed from and restored to isolate YB, or exchanged with those from other GRV isolates, showed that it carries the determinant for yellow blotch mosaic symptoms. Plants inoculated with the 0.9 kbp dsRNA (denatured or undenatured) developed yellow blotch mosaic even when the satellite-free GRV helper was not inoculated until 11 days later. The satellite RNA is therefore a very stable molecule. Prior infection of N. benthamiana with a GRV isolate containing a normal form of the satellite protected against expression of yellow blotch mosaic symptoms when the plants were later inoculated with isolate YB, whereas prior infection with satellite-free isolates did not. This provides a simple method of determining whether a GRV isolate has an associated satellite RNA. The YB satellite seems to be a newly recognised variant additional to those known to cause the chlorotic, green and other forms of groundnut rosette disease.     

含卫星RNA的黄瓜花叶病毒弱株系的分离鉴定及在病毒病防治上的应用

从豌豆上分离获得黄瓜花叶病毒分离物ＣＭＶＰ１,摩擦接种９科２９种植物,ＣＭＶＰｌ在大多数植物上症状很轻或无任何症状,提纯的病毒颗粒为球形,直径约２８ｎｍ,病毒衣壳蛋白亚基分子量约２７．５ｋＤ,所含核酸有五个组份,即ＣＭＶＰ１含有卫星ＲＮＡ。ＣＭＶＰ１接种三生烟后８－１２天内呈轻花叶,此时组织中病毒含量最高,随后症状消失,去除卫星ＲＮＡ能加重ＣＭＶＰ１在番茄上的症状,因而是卫星ＲＮＡ减轻了ＣＭＶＰｌ的病状。当ＣＭＶＰ１保护接种番茄后攻强毒,番茄发病率低,病情轻,保护率达９０％以上,并有一定的刺激生长作用,还能提早开花４天,植株结果数增多,成熟果实的颜色、形态、品质和重量均正常。ＣＭＶＰ１对烟草亦具有很好的保护效果。保护接种的植株能明显减少强毒株侵染,可减少９０％以上。     

Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.     

Role of satellite RNA of an Indian isolate of cucumber mosaic virus in inducing lethal necrosis of tobacco plants

Lethal necrosis or systemic stem necrosis followed by death of Nicotiana benthamiana, severe leaf deformations of N. tabacum cv. white burley and blister formations on N. tabacum cv. samsun NN symptoms were induced by experimental inoculations of CMV RNA preparations containing satellite RNA (sat-RNA). Inoculations of RNA preparations without sat-RNA did not induce that severe symptoms on these plants, only late mild mosaic was observed. It is suggested that sat-RNA of CMV isolate has a certain role for enhancing severity of symptoms in tobacco plants. Local and systemic lethal necrosis of N. benthamiana is due to sat-RNA present with genome of CMV isolate. It is the first report of lethal necrosis induced in N. benthamiana by CMV satellite.     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。