Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

A role for plant microtubules in the formation of transmission-specific inclusion bodies of Cauliflower mosaic virus

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.     

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     

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.     

Cauliflower mosaic virus promoters direct efficient expression of a bacterial G418 resistance gene in Schizosaccharomyces pombe

We have isolated and sequenced a cDNA clone which contains the entire coding sequence of the precursor to a subunit of wheat phosphoribulokinase (PRKase). (The enzyme is a homodimer). The cDNA contains 1533 bp and has an open reading frame of 1212 nucleotides. This encodes a protein with an amino-terminal transit sequence of 53 amino acids, while the part that forms the mature protein contains 351 amino acids and has a molecular weight of 39,200 daltons. A comparison of the wheat amino acid sequence with that already known for the mature protein of spinach reveals that there are identical residues in 86% of the positions but their transit peptides differ substantially from one another. The mature wheat and spinach proteins are identical in a segment of over 50 amino acids near the amino-terminus which is the region believed to be involved in ATP binding and in regulation by light of the catalytic activity of the enzyme. We further demonstrate that the expression of PRKase mRNA in wheat leaves is regulated in a developmental, tissue-specific and light dependent manner. We also show that the light-induced increase in the steady-state levels of this mRNA is dependent on the developmental stage of the leaf.     

甘蔗花叶病毒CP基因的高效表达和抗血清制备

将甘蔗花叶病毒的CP基因克隆到表达载体pET22b( ),并在大肠杆菌BL21(DE3)中得到高效表达,表达蛋白约占总蛋白的15％。用此蛋白制备了效价高专化性强的抗体。此方法可以解决制备马铃薯Y病毒属病毒的血清时遇到的病毒提纯产量低和寄主蛋白影响等问题。     

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.     

Conferring cadmium resistance to mature tobacco plants through metal-adsorbing particles of tomato mosaic virus vector

Tomato mosaic virus vectors were designed that produced, by a translational readthrough, a fusion protein consisting of coat protein and metal-binding peptide, as a result of which particles were expected to present the metal-binding peptides on their surface. When inoculated in plants, they were expected to replicate and form a metal-adsorbing artificial sink in the cytoplasm, so as to reduce metal toxicity. Vectors were constructed harbouring sequences encoding various lengths of polyhistidine as a metal-binding peptide. One of the vectors, TLRT6His, which contains a 6 x histidine sequence, moved systemically in tobacco plants, and its particles were shown to retain cadmium ions by an in vitro assay. When a toxic amount of cadmium was applied, the toxic effect was much reduced in TLRT6His-inoculated tobacco plants, probably as a result of cadmium adsorption by TLRT6His particles in the cytosol. This shows the possible use of an artificial sink for metal tolerance and the advantage of employing a plant viral vector for phytoremediation.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

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.     

The use of African cassava mosaic virus as a vector system for plants

This paper describes the development of a gene-displacement vector based on DNA1, one of two single stranded circular genomic components of a bipartite geminivirus, African cassava mosaic virus (ACMV). The DNA1 molecules of ACMV were cloned as dimers into a plant transformation vector and the constructs have been integrated into tobacco protoplasts by PEG-mediated DNA transfer. In transgenic plants extrachromosomal copies of DNA1 monomers could be detected. Deletion of the coat protein-encoding gene in chimeric constructs resulted in free DNA1 copies of reduced size, and extrachromosomal recombinant molecules were detected after displacement of the coat protein-encoding region by foreign DNA fragments of comparable size. Due to the absence of the second component of ACMV, DNA2, the transgenic plants are free from viral infection symptoms which allows the establishment of healthy transformants that carry a recombinant construct in an extrachromosomal form.     

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.     

外源基因在植物病毒表达载体中表达策略的研究进展

利用植物病毒表达载体表达外源蛋白是近年来发展起来的具有表达量大,速度快和廉价等优势的生产系统,其有4种构建策略;基因取代,基因插入,融合抗原和基因互补,此外还从病毒表达载体的基础性研究和商业应用方面进行了详细讨论。     

Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels

Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO₃ and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 × 10⁻¹² mol/L (3σ). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.