Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus／Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAβ, but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in N. benthamiana plants, although Y 132 DNA1 could replicate in these plants.     

A modified viral satellite DNA that suppresses gene expression in plants

DNAbeta is a type of single-stranded (ss) circular satellite DNA found in association with monopartite-genome begomoviruses, such as Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10). Y10 DNAbeta is required for symptom expression in plants but depends on TYLCCNV-Y10 genomic DNA (DNA-A) for replication and encapsidation. When we converted DNAbeta into a gene-silencing vector (modified DNAbeta (DNAmbeta)) by replacing its C1 open-reading frame (ORF) with a multiple cloning site (MCS), it was replicated but no longer induced symptoms in association with TYLCCNV-Y10 DNA-A, so allowing the effects of gene inserts to be recognized easily. Insertion into DNAmbeta of sequences from any of the three host genes (proliferating cell nuclear antigen (PCNA), phytoene desaturase (PDS), and sulfur (Su)), or from a transgene (green fluorescent protein (GFP)), resulted in silencing of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than a month and was associated with decreased levels of mRNA of the gene targeted. Although DNAmbeta probably does not enter meristematic tissue, the PCNA gene could be silenced there. DNAmbeta was an effective silencing vector in tested N. glutinosa, N. tabacum Samsun (NN or nn), and Lycopersicon esculentum plants, and was able to silence two genes simultaneously. This satellite DNA vector-based form of virus-induced gene silencing (VIGS) promises to be applicable to other begomovirus/DNAbeta systems, which are recently reported to occur in several dicotyledonous crop species, thereby providing a powerful approach to gene discovery and the analysis of gene function in these crops.     

Functional analysis of gene-silencing suppressors from tomato yellow leaf curl disease viruses

Tomato yellow leaf curl disease (TYLCD) is caused by a complex of phylogenetically related Begomovirus spp. that produce similar symptoms when they infect tomato plants but have different host ranges. In this work, we have evaluated the gene-silencing-suppression activity of C2, C4, and V2 viral proteins isolated from the four main TYLCD-causing strains in Spain in Nicotiana benthamiana. We observed varying degrees of local silencing suppression for each viral protein tested, with V2 proteins from all four viruses exhibiting the strongest suppression activity. None of the suppressors were able to avoid the spread of the systemic silencing, although most produced a delay. In order to test the silencing-suppression activity of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) proteins in a shared (tomato) and nonshared (bean) host, we established novel patch assays. Using these tools, we found that viral proteins from TYLCV were able to suppress silencing in both hosts, whereas TYLCSV proteins were only effective in tomato. This is the first time that viral suppressors from a complex of disease-causing geminiviruses have been subject to a comprehensive analysis using two economically important crop hosts, as well as the established N. benthamiana plant model.     

Molecular characterization of tomato leaf curl China virus, infecting tomato plants in China, and functional analyses of its associated betasatellite

A novel tomato-infecting begomovirus from Guangxi province, China, was identified and characterized, for which the name Tomato leaf curl China virus (ToLCCNV) was proposed. Phylogenetic and recombination analyses of the virus genomic sequences suggested that ToLCCNV may have arisen by recombination among Tomato leaf curl Vietnam virus (ToLCVV), Tomato leaf curl Gujarat virus (ToLCGV), and an unknown virus. A betasatellite molecule was found to be associated with ToLCCNV (ToLCCNB), and its complete nucleotide sequences were determined. Infectious clones of ToLCCNV and ToLCCNB were constructed and then used for agro-inoculation of plants; ToLCCNV alone infected Nicotiana benthamiana, Nicotiana glutinosa, Petunia hybrida, and Solanum lycopersicum plants, but no symptoms were induced. ToLCCNB was required for induction of leaf curl disease in these hosts. The βC1 protein of ToLCCNB was identified as a suppressor of RNA silencing and accumulated primarily in the nucleus. Deletion mutagenesis of βC1 showed that the central part of βC1 (amino acids 44 to 74) was responsible for both the suppressor activity and nuclear localization.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子

为了研究中国胜红蓟黄脉病毒(Ageratum yellow vein Chin virus,AYVCNV)和假马鞭曲叶病毒(Stachytarpheta leafcurl virus,StaLCV)C4蛋白的功能,利用烟草脆裂病毒(Tobacco rattle virus,TRV)载体在本氏烟(Nicotianabenthamiana)中分别表达了这两种病毒的C4蛋白,结果发现它们均能在本氏烟中引起类似于病毒侵染的症状,推测AYVCNV和StaLCV的C4蛋白是病毒的致病因子;在RNA沉默的抑制试验中,AYVCNV和StaLCV的C4蛋白均能够在表达gfp基因的转基因本氏烟(16c)上抑制由gfp基因正义链引起的基因沉默的建立,证明它们都是RNA沉默的抑制子。     

Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.     

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF βC1 in satellite DNA β which are predicted to function as silencing suppressors. In the present study suppressor function of ORF βC1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing βC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with βC1 gene from ToLCBB. However, in the case of 35S-βC1 CLCuMB and 35S-βC1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which βC1 interfere in RNA-silencing pathway is different.     

Long-distance movement,virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the gammab gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the gammab protein may be a long-distance movement factor and have antisilencing activity. This was shown for gammab proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, gammab and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the gammab cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus gammab proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.     

Phenotypes and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.     

Physiological function of NbRanBP1 in Nicotiana benthamiana

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.     

Agroinfection of sweet potato by vacuum infiltration of an infectious sweepovirus

Sweepovirus is an important monopartite begomovirus that infects plants of the genus Ipomoea worldwide. Development of artificial infection methods for sweepovirus using agroinoculation is a highly efficient means of studying infectivity in sweet potato. Unlike other begomoviruses, it has proven difficult to infect sweet potato plants with sweepoviruses using infectious clones. A novel sweepovirus, called Sweet potato leaf curl virus-Jiangsu (SPLCV-JS), was recently identified in China. In addition, the infectivity of the SPLCV-JS clone has been demonstrated in Nicotiana benthamiana. Here we describe the agroinfection of the sweet potato cultivar Xushu 22 with the SPLCV-JS infectious clone using vacuum infiltration. Yellowing symptoms were observed in newly emerged leaves. Molecular analysis confirmed successful inoculation by the detection of viral DNA. A synergistic effect of SPLCV-JS and the heterologous betasatellite DNA-β of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10) on enhanced symptom severity and viral DNA accumulation was confirmed. The development of a routine agroinoculation system in sweet potato with SPLCV-JS using vacuum infiltration should facilitate the molecular study of sweepovirus in this host and permit the evaluation of virus resistance of sweet potato plants in breeding programs.     

The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant

The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.