Expression Of Foreign Genes In Plants Research Articles

Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L−1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T 1 transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.

Characterization and comparative analysis of promoters from three plant pararetroviruses associated with Dahlia (Dahlia variabilis).

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV), and an endogenous plant pararetroviral sequence (DvEPRS, formerly known as DMV-D10) were reported from dahlia (Dahlia spp). Promoter elements from these dahlia-associated pararetroviruses were identified and characterized. The TATA box, the CAAT box, the transcription start site, the polyadenylation signal, and regulation factors, characteristic of caulimovirus promoters, were present in each of these promoter regions. Each of the promoter regions was separately cloned into a binary vector containing β-glucuronidase (GUS) reporter gene and delivered into Agrobacterium tumefaciens by electroporation followed by agroinfiltration into Nicotiana benthamiana. The activity of the 35S promoter homologs was determined by transient expression of the GUS gene both in qualitative and quantitative assays. The length of the promoter regions in DMV, DCMV, and DvEPRS corresponded to 438, 439, and 259 bp, respectively. Quantitative GUS assays showed that the promoters from DMV and DCMV resulted in higher levels of gene expression compared to that of DvEPRS in N. benthamiana leaf tissue. Significant differences were observed among the three promoters (p < 0.001). Qualitative GUS assays were consistent with quantitative GUS results. This study provides important information on new promoters for prospect applications as novel promoters for their potential use in foreign gene expression in plants.

A new agroinoculation technology for foreign gene expression in plants by means of transient expression

Due to the laborious and scale-up limitation we have developed a simple method named "seed absorption" to express foreign proteins in plants by means of transient expression. It has been shown that the reporter gene GFP was expressed successfully in tomato (Solanum lycopersicum L.) plants by seed absorbing Agrobacterium suspension containing TMV-based p35S-30B-GFP vector. Various factors influencing the gene expression were optimized including Agrobacterium cell density and other inoculation conditions. This method has the special advantages as simple work process, ease to scale-up, and further expanding the host range of plant bioreactor than previous methods. We assume that the seed absorption method will facilitate the industrial scale production of the recombinant pharmaceutical proteins in plants.

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

Improved foreign gene expression in plants using a virus‐encoded suppressor of RNA silencing modified to be developmentally harmless

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N.benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.

A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension

Due to the laborious and scale-up limitation we have developed a simple system named “root absorption” to express foreign proteins in plants successfully. It has been shown that GFP was expressed in tobacco plants by root absorbing the Agrobacterium suspension containing TMV-based P35S-30B-GFP vector. Various factors influencing the gene expression were studied including Agrobacterium cell density, seedling age, plant materials and inoculation conditions. This system has the special advantages as simple and convenient work process, ease to scale-up and higher level of expression than leaf infiltration. Interestingly, GFP was expressed at 24 h post-absorption. We assume that the root absorption system will facilitate the large-scale production of the recombinant pharmaceutical proteins in plants by means of transient expression.

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.

Improvement of the movement and host range properties of a plant virus vector through DNA shuffling.

Virus expression vectors based on the tobacco mosaic virus (TMV) genome are powerful tools for foreign gene expression in plants. However, the inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. To improve these properties of TMV vectors, the gene encoding the 30-kDa movement protein was subjected to mutagenesis and DNA shuffling. A vector that expresses the green fluorescent protein was used to allow simple visual discrimination of mutants with enhanced movement phenotypes. An initial round of mutagenesis produced 53 clones with a faster local movement phenotype. Two subsequent rounds of DNA shuffling produced additional clones that showed further increased rates of cell-to-cell movement and degrees of systemic invasion in restrictive hosts. Surprisingly, sequence analysis of the best performing shuffled genes revealed alterations resulting in coding and silent changes in the movement protein gene. Separation of these coding and silent alterations into distinct gene backgrounds revealed that each contributes to improved movement protein function to differing degrees. The resulting vectors demonstrate that the complex activities of the movement protein genes of viruses can be evolved to have improved movement phenotypes, as evidenced by cell-to-cell and systemic invasion. The experiments produced improved vectors that will be of use both for in planta functional screening and for therapeutic protein production and demonstrated the power of shuffling for plant virus vector improvement.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes.

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434-438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Transformation and foreign gene expression in plants by microprojectile bombardment.

AbstractMost common strategies for gene transfer in plants derive from the fact that Agrobacterium tumefaciens contains Ti plasmids which facilitate transfer of T-DNA from the plasmid genome into plant cells with attendant integration of the T-DNA region containing foreign genes into the plant nuclear genome (1). Alternatively, DNA may be introduced into protoplasts using various chemical fusagens (2), by electroporation (3), or by microinjection (4). In all these approaches foreign DNA is introduced into the nuclear genome and mostly single genes driven by independent promoters flanked by polyA-specifying terminators have been expressed. Despite several advantages of the Agrobacterium-mediated transformation, major limitations still remain; the limited host range should be extended to major crop species. Monocots, especially cereals, are highly recalcitrant to Agrobacterium infection. Since regeneration from cultured tissues is a prerequisite, problems of somaclonal variation resulting from tissue culture induced mutations should also be addressed. Direct DNA transfer by electroporation or using chemical fusagens require the use of protoplasts; unfortunately, most crops can not be regenerated from single cells (5).KeywordsChloroplast GenomeForeign GeneImmature EmbryoMultiple Cloning SiteTungsten ParticleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Replication of subterranean clover stunt virus in pea and subterranean clover protoplasts

Subterranean clover stunt virus (SCSV) was previously found to be representative of a new type of single-stranded DNA virus. Purified SCSV particles did not infect subterranean clover when various attempts were made to transmit them to this host using aphids ( Aphis craccivora) which had previously been fed on preparations of the virus. To demonstrate that purified preparations of SCSV are capable of replication, pea and subterranean clover protoplasts were inoculated with the virus using PEG treatment or electroporation, and maintained for up to 13 days. Up to 40% of the protoplasts survived after PEG treatment but less than 10% survived after electroporation. Both enzyme-linked immunosorbent assay (ELISA) and nucleic acid hybridisation detected de novo synthesis of SCSV in the inoculated protoplasts. The amount of SCSV coat protein detected by ELISA decreased from day 0 to day 3 post-inoculation but increased thereafter over several days to a maximum at about 10 days. Nucleic acid hybridisation studies using strand-specific probes showed that the kinetics of synthesis of the virion and its complementary strand nucleic acid corresponded with the kinetics of accumulation of SCSV antigens. These results suggest that purified SCSV particles are capable of replication in transient protoplast systems. The protoplast assay could be used to characterise new or altered segments or functional domains of the SCSV genome and for the development of SCSV genome as a vector for foreign gene expression in plants.

Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence

A synthetic transcribed, untranslated leader sequence from alfalfa mosaic virus RNA4 (AMV leader) has been assessed for its in vivo properties as a cis-active ‘translational activator’ in transient expression assays in protoplasts of Nicotiana tabacum and Picea glauca, as well as in stable expression in transformed Nicotiana tabacum. Levels of GUS enzyme activity produced by chimeric genes with or without the AMV leader sequence, in combination with either a CaMV 35S promoter or a duplicated-enhancer CaMV 35S promoter construct were assessed. In transient assay systems, the presence of a synthetic 40-base leader sequence lead to a 20-fold elevation in GUS activity when the constructs contained a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4-fold elevated expression lebel was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for translation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV constructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to assess relative transcript levels in plants expressing GUS from AMV-containing chimeric constructs. The results showed that elevated expression attributable to the AMV leader sequence was independent of abundance of the corresponding AMV- gus transcript, suggesting a post-transcriptional mechanism of action in vivo. Further, we describe the construction of general-purpose constitutive high expression plant promoter cassettes which incorporate the AMV translational enhancer sequence, as well a duplicated-enhancer 35S promoter in an optimized translational context.

Transformation of tomato using an Ri plasmid vector

An intermediate vector, pAMNeo10, was constructed containing the replication origin and carbenicillin-resistance gene of pBR322, an homology region to allow insertion into the T L-DNA of pRiA4 in Agrobacterium A4T, and a chimaeric kanamycin-resistance gene ( nop. neoΔ) for identification of T L-DNA::pAMNeo10 transformed roots. Roots produced by inoculating stem explants of Lycopersicon esculentum, L. hirsutum × L. esculentum (KNVF Rootstock) and L. peruvianum with an exconjugant stain, A4T (pRiA4::pAMNeo10), were resistant to kanamycin at levels that completely inhibit the growth of transformed roots produced with wild-type A4T. When transformed by the exconjugant strain, roots of the three tomato hosts were resistant to different levels of kanamycin, and, in the case of L. peruvianum, regenerated plants were tolerant to much higher levels (10×) of kanamycin than the transformed roots from which they were derived. Kanamycin-resistant transformed roots expressed aminoglycoside phosphotransferase activity, and Southern blotting confirmed the presence of the intermediate vector sequence in transformed roots and in shoots of regenerated plants. T R-DNA was shown to be present in most transformed roots and regenerated shoots by testing for agropine and mannopine. The application of Ri plasmid vectors to the study of foreign gene expression in plants is discussed.