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

We have developed a plant transient expression vector that simultaneously expresses -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.     

The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent

The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.     

A geminivirus AYVV-derived shuttle vector for tobacco BY2 cells

We have developed a plant-Escherichia coli pASV shuttle vector from the essential elements of the Ageratum yellow vein virus (AYVV). The geminivirus vector contains the AYVV genome with the coat-protein deletion, the E. coli vector backbone of pUC19, a unique cloning site and gene expression cassettes for plant selection and reporter gene activity. The replication of pASV vectors was compared in Nicotiana benthamiana and N. tabacum BY2 cells, and the latter were found to be suitable for long-term maintenance of the vectors in culture. The vector DNA was detected at regular intervals by PCR, -glucuronidase expression analysis and plasmid rescue during a 4-month culture period. A novel methylation-based PCR assay was carried out to show de novo replication for pASV-derived vectors in 2-month-old tobacco BY2 cell lines. This is the first report of the extrachromosomal replication of monopartite begomovirus with stability and foreign gene expression in long-term cell cultures.Abbreviations ACMV African cassava mosaic virus - AYVV Ageratum yellow vein virus - CP Coat protein - GUS -Glucuronidase - TGMV Tomato golden mosaic virus - Tobacco BY2 Tobacco L. cv. Bright Yellow 2     

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Production of virus resistant and insect tolerant transgenic tobacco plants

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r₀ and R₁ tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV cucumber mosaic virus - Bt toxin Bacillus thuringiensis -endotoxin - CaMV cauliflower mosaic virus - NOS nopaline synthase - Kan Kanamycin - Spe spectinomycin - Carb Carbenicillin     

Both TATA box and upstream regions are required for the nopaline synthase promoter activity in transformed tobacco cells

Summary Using a promoter expression vector system based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, we have studied the molecular structure of the nopaline synthase (nos) promoter which is active constitutively in transformed plant tissues. The system uses the sensitive and reliable chloramphenicol acetyltransferase (CAT) assay for the analysis of promoter strength in plant cells. Two sets of mutants were generated by sequential deletion of the nos promoter region from both 5 and 3 ends. These promoter fragments were linked to the cat coding sequence within the expression vector. The strength of the mutant promoters was measured in transformed tobacco calli as CAT activity. 3 deletions up to-17 bp did not significantly affect the promoter strength. Further deletions into the TATA box region reduced the promoter strength by about ten-fold. Analysis of the 5 deletion mutants showed that an upstream region is required for the nos promoter activity in addition to the TATA box and CCAAT box regions.     

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

Summary Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant court-noué disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a court-noué disease.Abbreviations GFLV-F13 grapevine fanleaf virus F13 isolate - ArMV arabis mosaic virus - CP coat protein - MS Murashige and Skoog - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - VPg genome linked viral protein - TMV tobacco mosaic virus - PVX potato virus X - PVY potato virus Y - TRV tobacco rattle virus - +CP CP expressing - -CP control plant, not expressing CP - CPMP coat protein-mediated protection - CPMCP coat crotein-mediated cross protection     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.