Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus

Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation. To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation. In this procedure, positive transformants could be clearly differentiated from the nontransformed plants. Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV). In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants. These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Expression of coat protein gene of Cucumber mosaic virus (CMV-subgroup IA) Gladiolus isolate in Nicotiana tabacum

The coat protein (CP)-mediated resistance against Cucumber mosaic virus (CMV) subgroup IA was developed in transgenic lines of Nicotiana tabacum cv. Petit Havana using Agrobacterium tumefaciens-mediated transformation. Ten independently transformed lines have developed, four of which were tested for resistance against CMV using virus challenge inoculations. The transgenic lines exhibiting complete resistance remained healthy and symptomless in their life span and showed reduced or no virus accumulation in their systemic leaves after virus challenge inoculation. These transgenic lines also showed resistance against CMV strains which are not closely related to CMV-Gladiolus strains. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IA member isolated from India showing resistance to all CMV strains occurring in the same vicinity.     

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.     

Variation between Turnip mosaic virus Isolates in Zhejiang Province,China and Evidence for Recombination

The 3′‐terminal sequences (c. 1700 nt) of the RNA genome of 10 Turnip mosaic virus (TuMV) isolates from different hosts in Zhejiang province, China, were determined. Phylogenetic analysis of the coat protein nucleotide sequences revealed that most TuMV sequences fell into two distinct clusters. The Chinese isolates B1‐B4 (from Brassica spp.) were similar and placed in the largest group (Group 1), while the isolates R1‐R6 (from Raphanus) were usually placed in a distinct but smaller group (Group 2). There were only approximately 90% identical nucleotides between the two groups. However, one isolate (R5) showed evidence of recombination in that the region between nucleotides 430 and 450, from the start of the coat protein gene and its 3′‐terminus, was a Group 1 type.     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

香蕉花叶病毒外壳蛋白基因克隆及表达载体的构建

从海南大田感染香蕉花叶病的香蕉叶片 ,获得香蕉花叶病毒 ,提纯其 RNA,在 AMV反转录酶作用下合成 c DNA第一链 ,经 PCR扩增 ,获得一约 70 0 bp的 DNA片段 ,测序结果显示所克隆的 DNA片段包含一完整的香蕉花叶病毒株系 ( CMV-BHI)外壳蛋白基因 ,长度为 6 5 7bp,然后将此 DNA片段 ,分别克隆到p BI1 2 1和 p KHG4质粒 ,构成两个含 Ca MV35 s启动子 ( 5 '-端 )、NOS终止子 ( 3'-端 )和分别含 NPT 标记基因和 NPT 及 HPT标记基因的植物表达载体 ( p TBB和 p TBK)。然后用 p AHC1 8中的 UBI promoter换下p BI1 2 1的 Ca MV35 s promoter,构成 p BIAH;再用 CMV-BHI外壳蛋白基因换下 p BIAH中 GUS基因 ,构成一含单子叶植物启动子 UBI和 NPT 标记基因的植物表达载体 ( p TBBU)。从而为 CMV-BHI外壳蛋白基因在香蕉中表达打下了基础     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

黄瓜花叶病毒基因组不同部分及卫星RNA介导的对植株的保护及其作用机理

根据病原物介导的对自身抗性的理论,大量开展了将CMV基因组的单个或多个片断转入植物体内的研究,从而使该植株能够抵抗或延迟受CMV的侵染,CP,RP,MP基因是CMV基因组的重要组成部分,用来转化植株取得了不同程度的抗性效果,另外有些CMV株中存在着起致弱作用的卫星RNA,直接对植株接种含卫星RNA的CMV弱毒或用卫星RNA的cDNA转化植株都会减轻CMV强毒对该植株的侵害,CMV基因组不同组分进入植物体内后,它们对植株产生保护作用的机理不同,文中分别加以阐述。     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

A Distinct Subpopulation of Cauliflower mosaic virus (CaMV) in South Iran

Cauliflower mosaic virus (CaMV) with a high incidence and widespread distribution on Brassica crops in Iran reduces the yield and quality of these crops. The complete sequences of three open reading frames (ORFs) 2, 4 and 6 coding for aphid transmission (AT), coat protein (CP) and inclusion body protein/translation transactivator (TAV) genes, respectively, were determined for two Iranian CaMV isolates from Kerman (south Iran). They induced latent or mild mottle (L/MMo) infection in Brassica oleracea var. capitata so are considered as the (L/MMo) biotype. Clear recombination breakpoints were detected between ORF2 and ORF6 in two Kerman isolates using concatenate fragments. Phylogenetic analysis revealed three Iranian CaMV subpopulations in which the two Kerman isolates in the new subgroup C were added to the two previously reported Iranian subpopulations A (central and west Iran) and B (north‐east Iran). Also three regions of pairwise identity were detected which representing: 97.1–100, 93.8–97.1 and 90.6–93.8% for subgroups A, C and B, respectively. Our analysis showed the high variability of Iranian CaMV population and provided valuable new information for understanding the diversity and evolution of caulimoviruses. Furthermore, star phylogeny was found in the subgroup C with overall lack of nt diversity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck although this may have been modified subsequently by clinal genetic drift. The appearance of new genetic types demonstrates a high potential of risks and should be considered in the planning of efficient control programmes.     

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

Transgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full-length CP, the core region of the CP, and full-length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full-length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene-encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.     

Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules

Structural changes in the single-stranded genome RNAs (RNAs 1, 2 and 3) and the subgenomic coat protein messenger (RNA 4) of alfalfa mosaic virus upon addition of a few coat protein molecules of the virus were investigated by measuring the fluorescent intensity of bound ethidium bromide and by circular dichroism. No effect could be observed in the case of the genome RNAs. However, in RNA 4, which is of much less complexity than the genome RNAs, a reduction of the ethidium bromide binding by 30% was found, whereas the positive molar ellipticity at 265 nm was reduced by 9% upon binding of the coat protein. Both changes point to a reduction of the ordered structure of the RNA. Since the protein is known to bind first at the 3′-terminus of RNA 4 and probably also of the genome RNAs, the conformational changes observed could be those thought to be necessary for replicase recognition in this positive-stranded RNA virus which needs the coat protein for starting an infection cycle.     

Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability

Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3′ untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 Å by X-ray fiber diffraction and refined it to an R-factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses, there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl-carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.