Showing papers on "Cauliflower mosaic virus published in 2003"
TL;DR: The generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter is described, indicating that an anti-L1 response was primed in about half of the 24 animals.
Abstract: Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Ω derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.
201 citations
TL;DR: It is demonstrated, that the compact 2-kb rbcS1 expression cassette provides a novel nuclear transformation vector that generates plants with expression levels of up to 10% of total protein.
Abstract: Transgenic plants are increasingly used as production platforms for various proteins, yet protein expression levels in the range of the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase have not yet been achieved by nuclear transformation. Suitable gene regulatory 5′ and 3′ elements are crucial to obtain adequate expression. In this study an abundantly transcribed member (rbcS1) of the ribulose-1,5-bisphosphate carboxylase small-subunit gene family of chrysanthemum (Chrysanthemum morifolium Ramat.) was cloned. The promoter of rbcS1 was found to be homologous to promoters of highly expressed rbcS gene members of the plant families Asteraceae, Fabaceae and Solanaceae. The regulatory 5′ and 3′ non-translated regions of rbcS1 were engineered to drive heterologous expression of various genes. In chrysanthemum, the homologous rbcS1 cassette resulted in a β-glucuronidase (gusA) accumulation of, at maximum, 0.88% of total soluble protein (population mean 0.17%). In tobacco (Nicotiana tabacum L.), the gusA expression reached 10% of total soluble protein. The population mean of 2.7% was found to be 7- to 8-fold higher than for the commonly used cauliflower mosaic virus (CaMV) 35S promoter (population mean 0.34%). RbcS1-driven expression of sea anemone equistatin in potato (Solanum tuberosum L.), and potato cystatin in tomato (Lycopersicon esculentum Mill.) yielded maximum levels of 3–7% of total soluble protein. The results demonstrate, that the compact 2-kb rbcS1 expression cassette provides a novel nuclear transformation vector that generates plants with expression levels of up to 10% of total protein.
136 citations
TL;DR: Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.
Abstract: The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.
118 citations
TL;DR: The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.
Abstract: The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.
116 citations
TL;DR: It is anticipated that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.
Abstract: Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.
113 citations
TL;DR: Cloned plant Bax Inhibitor-1 proteins cloned from cDNA libraries of oilseed rape and tobacco suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.
Abstract: To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants. Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells. As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.). The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%). Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains. Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression. Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter. All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein. Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER. Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.
91 citations
TL;DR: Transgenic plants ectopically expressingCsAGP1 showed a taller stature and earlier flowering than the wild-type plants and Y(βGlc)3, which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls.
Abstract: Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a "classical" arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(betaGlc)(3)-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(betaGlc)(3), which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation.
80 citations
TL;DR: Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter, indicating that the technique is a reliable measure of recombinant GFP expression.
Abstract: The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.
74 citations
TL;DR: Results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene.
Abstract: ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1. Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.
62 citations
TL;DR: The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.
Abstract: An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC = 6 dS m–1) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.
59 citations
TL;DR: Challenging these plants with bacterial or fungal phytopathogens demonstrated enhanced resistance up to the second generation, and transgenic plants displayed insecticidal properties.
Abstract: Esculentin-1 is a 46-residue antimicrobial peptide present in skin secretions of Rana esculenta. It is effective against a wide variety of micro-organisms, including plant pathogens with negligible effects on eukaryotic cells. As a possible approach to enhance plant resistance, a DNA coding for esculentin-1, with the substitution Met-28Leu, was fused at the C-terminal end of the leader sequence of endopolygalacturonase-inhibiting protein, under the control of the cauliflower mosaic virus 35S promoter region, and introduced into Nicotiana tabacum. The antimicrobial peptide was isolated from the intercellular fluids of healthy leaves of transgenic plants, suggesting that it was properly processed, secreted outside cells and accumulated in the intercellular spaces. The morphology of transgenic plants was unaffected. Challenging these plants with bacterial or fungal phytopathogens demonstrated enhanced resistance up to the second generation. Moreover, transgenic plants displayed insecticidal properties.
TL;DR: Histochemical staining for GUS activity on transgenic tobacco plants has further indicated a preferential expression in vascular tissues of stems and leaves conferred by the positive regulatory region.
Abstract: The genomic sequence of a rice (Oryza sativa L.) glycine-rich protein (GRP) gene, designated Osgrp-2, has been previously determined (GenBank U40708). Primer extension analysis indicated that transcription starts 47 bp upstream of the translation start codon. To gain an insight into the transcriptional regulation of this gene, the 2,401-bp promoter sequence and a series of its 5′ deletions were transcriptionally fused to the β-glucuronidase (GUS) gene. GUS activity was subsequently assayed in a transient expression system of tobacco (Nicotiana tabacum L.) protoplasts, which revealed the presence of a positive regulatory region (–2290 to –1406) and two negative regulatory regions (–2401 to –2291 and –1405 to –1022) in the Osgrp-2 promoter for the promoter activity. The positive regulatory region displayed an enhancer-like activity when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter (–89 to +6) to drive GUS expression and assayed on tobacco leaves by the Agrobacterium-mediated transient expression technique (agroinfiltration). Histochemical staining for GUS activity on transgenic tobacco plants has further indicated a preferential expression in vascular tissues of stems and leaves conferred by the positive regulatory region. A 1,023-bp fragment of the Osgrp-2 promoter (–1021 to +2) fused with GUS was transformed into tobacco and proved to be capable of conferring vascular-specific expression. Further 5′ and 3′ deletion analysis of the 1,023-bp promoter revealed that a 99-bp fragment located from –497 to –399 contained cis-elements responsible for vascular-specific expression.
TL;DR: Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects and could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein.
Abstract: A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.
TL;DR: These findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR- 1a as-1-like element.
Abstract: Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.
TL;DR: Results from this study indicate that RUBQ2 can serve as a new regulatory element to provide high levels of transgene expression in sugarcane through particle bombardment and Agrobacterium-mediated transformation.
TL;DR: Tomato plants were transformed with gene constructs that contained the gdhA gene for NADP-dependent glutamate dehydrogenase from Aspergillus nidulans coupled in the sense orientation with the constitutively active 35S promoter from cauliflower mosaic virus.
TL;DR: Results indicate that CaMV basic replication requires both Tav-activated polycistronic translation and some posttranslational function(s) of Tav that is not affected by the deletions in the central region of Tav.
Abstract: Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is an essential multifunctional viral protein. Dissection of Tav by deletion mutagenesis revealed that the central region is essential for CaMV replication in single cells but that the N- and C-terminal parts are not. Strains with mutations in the central region were defective in the translational transactivator function and could be complemented by coexpressing Gag (capsid protein precursor) and Pol (polyprotein with protease, reverse transcriptase, and RNase H activity) from separate monocistronic plasmids. In contrast, total omission of Tav was only partially complemented by Gag and Pol overexpression from separate plasmids. These results indicate that CaMV basic replication requires both Tav-activated polycistronic translation and some posttranslational function(s) of Tav that is not affected by the deletions in the central region of Tav.
TL;DR: The results support the notion that in a large proportion of the T‐ DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T‐DNA integrations.
TL;DR: The occurrence of BWYV, TuMV and CaMV in oilseed rape crops could have important consequences for rape seed production in Iran and infected plants could also serve as sources of virus for other susceptible vegetable crops.
Abstract: Oilseed rape (Brassica napus ssp. oleifera) is an important crop in Iran. Of 581 oilseed rape samples tested by DAS-ELISA and immunoelectron microscopy during a preliminary survey from 1999 to 2001, 14.45% were infected with Beet western yellows virus (BWYV, Luteovirus), 12.9% with Cauliflower mosaic virus (CaMV, Caulimovirus) and 9.3% with Turnip mosaic virus (TuMV, Potyvirus). Cucumber mosaic virus (4.6%) and Tomato spotted wilt virus (0.51%) also occurred, but to a lesser extent. The occurrence of BWYV, TuMV and CaMV in oilseed rape crops could have important consequences for rape seed production in Iran and infected plants could also serve as sources of virus for other susceptible vegetable crops.
TL;DR: The iris RIP is an interesting and useful model for the study and exploitation of the antiviral activity of type-1 RIPs and shows no apparent phenotypic side effects indicating that ectopically expressed IRIP is not cytotoxic for tobacco cells.
Abstract: To study the in planta antiviral activity of a type-1 ribosome-inactivating protein from iris bulbs, called IRIP,Nicotiana tabacumcv. Samsun NN was transformed with the IRIP sequence expressed under the control of the 35S cauliflower mosaic virus promoter. Molecular analysis of the transgenic plants and characterization of the purified protein revealed that the recombinant IRIP from tobacco leaves has the same molecular structure and RNA N-glycosidase activity as the native protein from iris bulbs. The tobacco transformants show no apparent phenotypic side effects indicating that ectopically expressed IRIP is not cytotoxic for tobacco cells. No induction of PR-1 could be demonstrated in the transgenic plants expressing IRIP. The in planta antiviral activity of rIRIP was assessed using a bioassay with tobacco mosaic virus. All transformed lines showed a statistically significant lower number of lesions compared to the control plants. The fortunate combination of in planta antiviral activity and lack of cytotoxicity of the ectopically expressed IRIP in transgenic tobacco renders the iris RIP an interesting and useful model for the study and exploitation of the antiviral activity of type-1 RIPs.
TL;DR: Data show that, although the str246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-responsiveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.
Abstract: In order to improve pear resistance against fire blight caused by Erwinia amylovora, a search for promoters driving high-level expression of transgenes specifically in response to this bacterial pathogen has been undertaken. We have examined the ability of hsr203J, str246C and sgd24 tobacco (Nicotiana tabacum L.) promoters to drive expression of the uidA reporter gene in pear. Transgenic pear clones were obtained by Agrobacterium tumefaciens-mediated transformation. β-Glucuronidase activity was determined quantitatively and qualitatively in these plants grown in vitro using fluorometric and histochemical assays and compared to cauliflower mosaic virus (CaMV) 35S promoter-driven activity. The hsr203J promoter appeared to be very weakly activated following inoculation in pear, which is the converse of the situation in tobacco. The str246C promoter was rapidly activated in pear during compatible and incompatible interactions, by wounding and following the application of several elicitors (capsicein, cryptogein, harpin, salicylic acid and jasmonic acid). The sgd24 promoter, a deletion derivative of str246C, exhibited a low level of expression after bacterial inoculation, was weakly activated by wounding and elicitors, and was not activated by phytohormones (salicylic acid and jasmonic acid). Interestingly, the sgd24 promoter was locally activated in pear, whereas the str246C promoter was activated systemically from the infection site. Taken together, these data show that, although the str246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-responsiveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.
01 Jan 2003
TL;DR: In this article, the ability of /wr203J, str246C and sgd24 tobacco promoters to drive expression of the uidA reporter gene in pear has been examined in order to improve pear resistance against fire blight caused by Erwinia amylovora.
Abstract: In order to improve pear resistance against fire blight caused by Erwinia amylovora, a search for pro moters driving high-level expression of transgenes spe cifically in response to this bacterial pathogen has been undertaken. We have examined the ability of /wr203J, str246C and sgd24 tobacco (Nicotiana tabacum L.) promoters to drive expression of the uidA reporter gene in pear. Transgenic pear clones were obtained by Agrobacterium tumefaciens-mediated transformation. ^-Glucuronidase activity was determined quantitatively and qualitatively in these plants grown in vitro using fiuorometric and histochemical assays and compared to cauliflower mosaic virus (CaMV) 35S promoter-driven activity. The hsr203J promoter appeared to be very weakly activated following inoculation in pear, which is the converse of the situation in tobacco. The str246C promoter was rapidly activated in pear during compat ible and incompatible interactions, by wounding and following the application of several elicitors (capsicein, cryptogein, harpin, salicylic acid and jasmonic acid). The sgd24 promoter, a deletion derivative of str246C, exhibited a low level of expression after bacterial inoc ulation, was weakly activated by wounding and elicitors, and was not activated by phytohormones (salicylic acid and jasmonic acid). Interestingly, the sgd24 promoter was locally activated in pear, whereas the str246C pro moter was activated systemically from the infection site. Taken together, these data show that, although the s/r246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-respon siveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.
TL;DR: Constitutively over-expressed RARFI under the control of the cauliflower mosaic virus 35S promoter triggered spontaneous induction of lesion mimics, induced an array of pathogenosis-related (PR) genes, reduced susceptibility to a fungal pathogen, and caused accumulation of SA.
Abstract: A cDNA encoding RARF1 (rice ADP-ribosylation factor 1) was isolated from fungal elicitor-treated rice suspension culture cells by mRNA differential display. RARF1 transcripts accumulated in response to hydrogen peroxide (H 2 O 2 ) and salicylic acid (SA) and rapidly in cells inoculated with an avirulent pathogen. Constitutively over-expressed RARFI under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) triggered spontaneous induction of lesion mimics, induced an array of pathogenosis-related (PR) genes, reduced susceptibility to a fungal pathogen, and caused accumulation of SA. From these data, we deduced that RARF1 might be a component of various plant defence signalling pathways involved in inducing the expression of a subset of PR genes.
TL;DR: An unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana is characterized and comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line, indicates that the TAVfunction of P 6 may play only a minor role in the development of chlorotic symptoms.
Abstract: The gene VI protein (P6) of Cauliflower mosaic virus (CaMV) functions as a virulence factor in crucifers by eliciting chlorotic symptoms in infected plants. The ability to induce chlorosis has been associated previously with P6 through gene-swapping experiments between strains and through the development of transgenic plants that express P6. The primary role that has been identified for P6 in the CaMV infection cycle is to modify the host translation machinery to facilitate the translation of the polycistronic CaMV 35S RNA. This function for P6 has been designated as the translational transactivator (TAV) function. In the present study, we have characterized an unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana. The level of D4 P6 produced in transgenic Arabidopsis line D4-2 was comparable to the amount found in transgenic plants homozygous for W260 and CM1841 P6, two versions of P6 that induce strong chlorotic symptoms and stunting in Arabidopsis. A complementation assay proved that P6 expressed in the D4-2 line was functional, as it could support the systemic infection of a CM1841 mutant that contained a lethal frame-shift mutation within gene VI. This complementation assay allowed us to separately assess the contribution of CM1841 gene VI to symptom development versus the contribution of other CM1841 genes. Furthermore, a previous study had shown that the TAV activity of D4 P6 was comparable to that of W260 P6. That comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line in the present paper, indicates that the TAV function of P6 may play only a minor role in the development of chlorotic symptoms.
TL;DR: During the spring and summer of 2001, surveys of 50 plantings of vegetable brassicas and associated wild radish and associatedWild radish weeds were done to determine the occurrence of three aphid-borne viruses, Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV) and Turnip mosaicirus (TuMV).
Abstract: During the spring and summer of 2001, surveys of 50 plantings of vegetable brassicas (broccoli, cabbage and cauliflower) and associated wild radish (Raphanus raphanistrum) weeds were done to determine the occurrence of three aphid-borne viruses, Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV) and Turnip mosaic virus (TuMV). Plantings at 15 widely separated farms were sampled in the Perth metropolitan and Manjimup regions of south-west Australia. None of the three viruses was detected in vegetable brassica plantings at Manjimup and neither were CaMV or TuMV detected in the Perth metropolitan region. BWYV was only detected at a low incidence (2% infection) in one planting of cabbage at Carabooda. In contrast, populations of wild radish from one farm in Perth and five out of six farms at Manjimup were infected with BWYV at incidences up to 65%. One population of wild radish was also infected with TuMV at an incidence of 2%. Additional keywords : Cauliflower mosaic virus, Turnip mosaic virus, Beet western yellows virus, surveys, virus reservoirs, control.
TL;DR: The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.
Abstract: To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f 9 coding for spidroin 1 analogs were 3"-fused in-frame with the reporter lichenase gene. The Tr2" weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.
TL;DR: Alfalfa and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea to demonstrate that PsUGT1 expression occurs in regions of active cell division.
Abstract: Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.
Journal Article•
TL;DR: It is suggested that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.
Abstract: The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.
TL;DR: Two distinct domains of the xynA gene from Clostridium thermocellum encoding a xylanase catalytic domain (XynAl) and a XynA2 under the control of the cauliflower mosaic virus 35S promoter were electroporated into cultured tobacco BY-2 cells.
TL;DR: In this paper, transient expression of several constructs containing β-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells.