Figwort Mosaic Virus Research Articles

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia.

Alpha-galactosidase (alpha-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the alpha-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of alpha-Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that alpha-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of alpha-Gal mRNAs. alpha-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from -4 degrees C for cold-acclimated wild-type plants to -8 degrees C for the most tolerant antisense line. Down-regulating alpha-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the alpha-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with alpha-Gal provides an additional method for improving the freezing tolerance of plants.

Partial Molecular Characterization of Dahlia mosaic virus and Its Detection by PCR.

Dahlia mosaic virus (DMV) is the causal agent of one of the most important diseases of Dahlia pinnata. The nucleotide sequence of a 1,195-bp fragment of its genome was amplified and characterized. Based on this sequence, polymerase chain reaction (PCR) assays were developed for detection of DMV. The nucleotide sequence confirmed the classification of DMV as a member of genus Caulimovirus since it was similar to a region covering partly open reading frames (ORFs) IV and V found in caulimoviruses. The two most closely related viruses on the basis of comparison of ORF V fragments were shown to be Figwort mosaic virus and Mirabilis mosaic virus with 66.6 and 68.1% identity, respectively. Two PCR assays were developed using identical primer pairs: a real-time PCR based on SYBR green chemistry and a conventional PCR. Both methods clearly discriminated DMV-infected and healthy dahlia. The real-time PCR assay detected DMV-infected material that was diluted 105-fold in healthy material.

Assessment of screening methods for the identification of genetically modified potatoes in raw materials and finished products.

Qualitative polymerase chain reaction methods for the detection of genetically modified potatoes have been investigated that can be used for screening purposes and identification of insect-resistant and virus-resistant potatoes in food. The presence of the nos terminator from Agrobacterium tumefaciens and the antibiotic marker gene nptII (neomycin-phosphotransferase II) was demonstrated in three commercialized Bt-potato lines (Monsanto Co., St. Louis, MO, USA) and one noncommercial GM-potato product (high amylopectin starch, AVEBE, Veendam, The Netherlands) and allows for general screening in foods. For further identification, specific primers for the FMV promoter derived from the figwort mosaic virus, the CryIIIA gene (delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis), potato leafroll virus replicase gene, and the potato virus Y coat protein gene, were designed. The methods described were successfully applied to processed potato raw materials (dehydrated potato powders and flakes), starch samples, and finished products.

Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the cauliflower mosaic virus promoters in monocot and dicot cells

A sub-genomic transcript (Sgt) promoter was isolated from the Figwort mosaic virus (FMV) genomic clone. The FMV Sgt promoter was linked to heterologous coding sequences to form a chimeric gene construct. The 5′-3′-boundaries required for maximal activity and involvement of cis-sequences for optimal expression in plants were defined by 5′-, 3′-end deletion and internal deletion analysis of FMV Sgt promoter fragments coupled with a β-glucuronidase reporter gene in both transient protoplast expression experiments and in transgenic plants. A 301 bp FMV Sgt promoter fragment (sequence −270 to +31 from the transcription start site; TSS) provided maximum promoter activity. The TSS of the FMV Sgt promoter was determined by primer extension analysis using total RNA from transgenic plants developed for FMV Sgt promoter: uidA fusion gene. An activator domain located upstream of the TATA box at −70 to −100 from TSS is absolutely required for promoter activity and its function is critically position-dependent with respect to TATA box. Two sequence motifs AGATTTTAAT (coordinates −100 to −91) and GTAAGCGC (coordinates −80 to −73) were found to be essential for promoter activity. The FMV Sgt promoter is less active in monocot cells; FMV Sgt promoter expression level was about 27.5-fold higher in tobacco cells compared to that in maize cells. Comparative expression analysis of FMV Sgt promoter with cauliflower mosaic virus (CaMV) 35S promoter showed that the FMV Sgt promoter is about 2-fold stronger than the CaMV 35S promoter. The FMV Sgt promoter is a constitutive promoter; expression level in seedlings was in the order: root>leaf>stem.

Agrobacterium -mediated transformation of the commercially important grapefruit cultivar Rio Red ( Citrus paradisi Macf.)

Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing β-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis.

Synthesis of Figwort Mosaic Virus (FMV) Coat Protein-Reverse Transcriptase Fusion Protein in Reticulocyte Lysates from a Full-length Transcript

Genetic organization of figwort mosaic virus (FMV) genome (circular, double stranded DNA of about 8 kbp) reveals six major open reading frames (I-VI) and a small (VII) open reading frame (ORF) which are expressed from two different transcripts. One of the transcripts spans the entire length of the viral genome and may function as a messenger RNA for the translation of 5-6 closely spaced genes appearing on this RNA; the other spans the gene VI. In the present study, synthesis of a native viral polypeptide(s) was investigated from a full-length and a partial transcript of a naturally occurring deletion mutant of FMV. The deletion in the mutant results in the fusion of the coat protein (IV) and the reverse transcriptase (V) cistrons, consequently, a fusion cistron (IV/V ORF) is generated. In constructs pH52 and pH55 used in the present study, chloramphenicol acetyltransferase (CAT) gene in the deletion mutant is positioned in cistron II and VI, respectively. Translation of full-length transcripts generated from these constructs in reticulocyte Iysates results in simultaneous synthesis of CAT polypeptide with a predicted size of the fusion (IV/V) polypeptide of 86,000 daltons. These polypeptides are immunoselected using anti-CAT and antiviral coat protein antibodies. When IV/V ORF is deleted from these constructs by digestion with Sac I before a transcript is prepared, with pH55, the in vitro expression of the fusion polypeptide (IV/V) and the CAT polypeptide in Iysates are both abolished. With construct pH52, on the other hand, the synthesis of CAT polypeptide is still observed. These studies are consistant with the full-length viral RNA functioning as a messenger RNA.

Mapping Regions of the Cauliflower Mosaic Virus ORF III Product Required for Infectivity

The open reading frame (ORF) III product (PIII) of the pararetrovirus cauliflower mosaic virus (CaMV) has nucleic acid-binding propertiesin vitro,but its biological role is not yet determined. ORF III is closely linked to ORF II and overlaps ORF IV out of frame in the CaMV genome. A new CaMV-derived vector (CaΔ) devoid of ORF III and containing unique restriction sites between ORFs II and IV was designed. Introduction of the wild-type CaMV ORF III into CaΔ results in a clone (Ca3) infectious in turnip plants. Truncated or point-mutated versions of ORF III were then inserted into CaΔ and testedin vivo.Inoculation of the different mutants into turnip revealed that the four C-terminal amino acid residues of PIII are dispensable for infectivity as well as an internal domain (amino acids 61 to 80). Taken together the results show that PIII possesses a functional two-domain organization. Moreover, the CaMV PIII function(s) cannot be replaced either by the PIII protein of another caulimovirus, the figwort mosaic virus, or by the P2 protein of the cacao swollen shoot badnavirus, a member of the second plant pararetrovirus group.

Isolation and Expression Analysis of Peanut Chlorotic Streak Caulimovirus (PClSV) Full-Length Transcript (FLt) Promoter in Transgenic Plants

A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

Strawberry vein banding virus--definitive member of the genus Caulimovirus.

The complete DNA sequence (7876 nucleotides) of strawberry vein banding virus (SVBV) has been determined. Seven open reading frames are detected that potentially code for proteins of calculated weight 37.8; 18.3; 16.6; 56.0; 81.1; 59.0 and 12.6 kDa, respectively. Their position on the viral genome is the same as that of the corresponding proteins on the cauliflower mosaic virus (CaMV) genome. Phylogenetic analysis based on the amino acid sequence of this protein shows a closer relationship of SVBV with CaMV, figwort mosaic virus and carnation etched ring virus than with other caulimoviruses.

Construction of a Plant Transient Expression Vector which Coexpresses the Marker β-glucuronidase

We have developed a plant transient expression vector that simultaneously expresses β-glucuronidase from a Cauliflower mosaic virus promoter, and a test gene from a Figwort mosaic virus promoter. This vector, which is manipulated in E. coli, allows the testing of cell death inducing genes in plant cells. We have demonstrated the capability of this vector by expressing diphtheria toxin.

Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

The Computer Simulation of RNA Folding Pathways Using a Genetic Algorithm

A procedure for simulating the RNA folding process using the principles of genetic algorithm is proposed. The method allows one to simulate a folding pathway of RNA, including such processes as disruption of temporarily formed structures, the folding of a molecule during its synthesis and pseudoknot formation. The simulations are able to predict functional metastable foldings and kinetically driven transitions to more stable structures. The analysis of free energies for intermediate foldings allows estimation of the ranges of kinetic refolding barriers and suggests that in some RNAs the selective evolutionary pressure suppresses the possibilities for alternative structures that could form in the course of transcription. It is shown that the folding pathway simulation can result in structure predictions that are more consistent with phylogenetically proven structures than minimum energy solutions. This suggest that suggest that RNA folding kinetics is very important for the formation of functional RNA structures. Therefore, apart from its value for predictions RNA structures, the proposed computer stimulations can be a powerful tool in the studies of RNA folding features.f2f2Abbreviations used: GA, genetic algorithm; STNV, satellite tobacco necrosis virus; UTR, untranslated region; CaMV, cauliflower mosaic virus; CERV, carnation etched ring virus; FMV, figwort mosaic virus; MNV11, minivariant; SV11, salt-resistant variant.

Systemic Infection of Solanaceous Hosts by Peanut Chlorotic Streak Caulimovirus Is Temperature Dependent and Can Be Complemented by Coinfection with Figwort Mosaic Caulimovirus

Peanut chlorotic streak caulimovirus (PClSV) developed systemically in tobacco (Nicotiana tabacum) and Datura inoxia at 33 C but not at 25 C even though the virus multiplied and was highly productive in the inoculated leaves of these plants at 25 C. In contrast to behavior in these hosts, the virus caused productive systemic infections at either temperature in Datura stramonium. PClSV was complemented for systemic infection in D. inoxia at 25 C by coinfection with a second caulimovirus, figwort mosaic virus. Gene I (tentative movement function) mutants of PClSV were not complemented for systemic development in D. inoxia at 25 C, suggesting that some other virus function was lacking for systemic development in tobacco and D. inoxia at 25 C

Analysis of Figwort Mosaic Virus (Plant Pararetrovirus) Polyadenylation Signal

Analysis of the cauliflower mosaic virus (CaMV) polyadenylation (poly(A)) signal has revealed several striking differences to poly(A) signals from animal genes such as the absence of activating sequences downstream from the cleavage site. Instead, upstream sequences were shown to induce recognition of an AAUAAA sequence. To test whether these features are representative of other plant pararetrovirus poly(A) signals, a characterization of the figwort mosaic virus (FMV) poly(A) signal is presented here. The FMV RNAs were isolated from infected plants and mapped, and the different elements composing the FMV poly(A) signal were identified. Multiple upstream sequences were found to be essential for efficient processing at the FMV poly(A) site and could be replaced by the CaMV upstream elements. The FMV upstream sequences showed homologies to other characterized upstream sequences from CaMV, from animal viruses, and from plant poly(A) signals. Surprisingly, neither the FMV nor the CaMV upstream elements could induce recognition of an AAUAAA sequence present in the FMV poly(A) signal, instead a UAUAAA sequence 55 nucleotides further downstream was utilized. It is proposed that additional features may be required for appropriate cleavage such as the context of the AAUAAA-like sequence or perhaps the cleavage site itself.

An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice

The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA. The complete sequence of a cloned infective ScBV genome is reported here. The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus. A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present. The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K. The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements. In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses. Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved. Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses. When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster. The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters. When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana.

Regulation of caulimovirus gene expression and the involvement of cis-acting elements on both viral transcripts

In a further analysis of gene regulation of figwort mosaic virus (FMV), a caulimovirus, we studied transient gene expression with modified viral genomes in Nicotiana edwardsonii cell suspension protoplasts. The results demonstrated that the presence of the promoter for the full-length RNA interferes with expression from the separate downstream promoter for gene VI. In addition, expression of gene VI was inhibited by cis-acting sequences within gene VI itself. Both inhibitory effects could be partially relieved by coelectroporation with a plasmid that produces gene VI protein, demonstrating that expression of gene VI is transactivated by its own product. Subsequent expression studies with partially redundant FMV plasmids containing a reporter gene in frame with gene IV showed that efficient transactivation of CAT expression relies on a cis-acting element inside the downstream gene VI. Insertions of a transcriptional terminator upstream of the cis-acting element for premature termination of transcription showed that the cis-acting region is not a DNA element but is active only as a feature of the RNA transcript. We conclude that the cis-acting element, together with the transacting gene VI product, enhances expression of all major genes, including gene VI, from the polycistronic m RNA and the separate m RNA for gene VI.

The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

Requirement of gene VII in cis for the expression of downstream genes on the major transcript of figwort mosaic virus

The six major conserved genes of figwort mosaic virus (FMV), a caulimovirus, appear in tandem array on an RNA transcript that spans the entire viral genome. Gene VI, the only cistron that appears as a separate subgenomic RNA, has been reported to transactivate the expression of downstream genes of the full-length transcript. This transcript has a long 5′-leader of about 600 nucleotides followed by a small nonconserved region (gene VII), a smaller intergenic region (57 nucleotides), and the major conserved genes in a closely spaced array. In our present experiments we have constructed expression units containing the promoter for the full-length transcript followed by the 5′ leader region, gene VII, and a reporter gene. These have been tested for expression with and without gene VI as a separate plasmid by electroporation into plant protoplasts. A series of these expression units containing truncated versions of the 5′ leader region placed upstream of a reporter gene (CAT) showed that gene VI transactivation occurred only when gene VII sequences were present in cis between the leader region and the reporter gene. In addition, a more complete version of the FMV genome containing the reporter gene further downstream (in viral gene IV) showed CAT expression only when gene VII sequences were present in an upstream position. A similar construct failed to express CAT activity when gene VII was absent.