Double Cauliflower Mosaic Virus 35S Research Articles

Tolerance of Arabidopsis thaliana plants overexpressing grapevine VaSTS1 or VaSTS7 genes to cold, heat, drought, salinity, and ultraviolet irradiation

Stilbene synthases (STS) are plant enzymes that are responsible for the biosynthesis of stilbenes, which are plant phenolic compounds with valuable biological properties. Stilbenes also play important roles in plant tolerance to biotic and abiotic stresses. Therefore, plants that overexpress STS genes can be more resistant to various stresses. This paper investigated the effects of STS gene overexpression in Arabidopsis thaliana (L.) Heynh. Columbia-0 plants on stilbene content and tolerance to the following abiotic stresses: low and high temperatures, salinity, drought, and ultraviolet irradiation (UV-B and UV-C). We used VaSTS1 and VaSTS7 genes from grapevine (Vitis amurensis Rupr.) expressed under the double cauliflower mosaic virus 35S (CaMV35S) promoter. This study firstly demonstrated that overexpression of the VaSTS1 and VaSTS7 genes in A. thaliana plants considerably increased plant tolerance to UV-B and UV-C, while the tolerance to the low and high temperatures, salinity, and drought was not affected. We showed that the highest trans-piceid and trans-resveratrol total content was in ST1 A. thaliana plants that overexpressed the VaSTS1 gene in the range 8.28 - 22.66 µg g-1(f.m.). ST7 plants that overexpressed the VaSTS7 gene showed only trans-resveratrol at 0.02 - 0.08 µg g-1(f.m). Stilbene content and UV tolerance in transgenic A. thaliana plants correlated with STS transgene expression. STS expression, UV tolerance, and stilbene content was higher in VaSTS1 transgenic plants compared with that in VaSTS7 transgenic plants.

35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress.

The cauliflower mosaic virus (CaMV) 35S promoter is known as the most frequently used promoter in plant biotechnology. Although it is widely considered to be a strong constitutive promoter exhibiting high transcriptional activity, the transcriptional stability of CaMV 35S has not been extensively studied. Using the model plant species Arabidopsis thaliana, this study aimed for a comprehensive expression analysis of two widely used plant transgenes, neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (EGFP), regulated by a double CaMV 35S promoter depending on the organ type, time of day, plant age, and in response to abiotic stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the NPTII and EGFP transcript levels were markedly higher in the cotyledons, young leaves, and roots than in the inflorescences, stems, and adult leaves of three independent transgenic A. thaliana lines. The expression of NPTII and EGFP varied during the day and was elevated with the plant age. Drought and cold stress considerably affected the expression of the transgenes, while heat, high salinity, and wounding had no significant effect. This study shows that transgenes driven by a common constitutive promoter can exhibit marked variations in transcriptional activity depending on plant organ, physiological conditions, and in response to abiotic stress. Therefore, to ensure high and stable transgene activity, considerable attention should be given to the transgenic plant material and incubation conditions before harvesting the plant material.

Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants. Two commonly used promoters, double cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS), and one regulator, tobacco etch virus (TEV) translational enhancer, were tested. Four different promoter/enhancer combinations were constructed to drive a bialaphos resistance gene (bar) and used for multiple independent transformation experiments. Herbicide application and PCR analysis were used to confirm the inheritance of the bar gene in the T 1 generation. Quantitative real-time PCR (qRT-PCR) was used to estimate transgene copy number in T 1 transgenic events. The average transformation frequency from combining NOS with the TEV enhancer (3.5% across 12 replications) was significantly higher than the frequencies obtained from the double CaMV 35S without an enhancer (1.6% across 16 replications), double CaMV 35S with an enhancer (1.4% across 8 replications), and NOS without an enhancer (1.0% across 12 replications). The bar transcript levels in T 1 transgenic leaf tissue did not correlate with transformation frequencies achieved by the different constructs. No significant differences were identified between constructs in the average transgene copy number across events. These data show that a selectable marker system comprised of the bar gene regulated by the NOS promoter in combination with the TEV enhancer is preferred in Agrobacterium-mediated soybean transformation.

Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean.

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions.

Overexpression of the oil palm (Elaeis guineensis Jacq.) TAPETUM DEVELOPMENT1-like Eg707 in rice affects cell division and differentiation and reduces fertility

The functional analysis of the TAPETUM DEVELOPMENT1-like analog Eg707 of oil palm was carried out in rice by over-expressing Eg707 under the control of a double cauliflower mosaic virus 35S promoter. Ectopic expression of Eg707 in rice induced dark green and matured compact brownish calli compared to pale wild type and negative control calli. Regenerated transgenic rice plants exhibited a reduction in organ size and plant height, rolled, erect leaves, less tillers, increased chlorophyll content, and reduced fertility with smaller green seeds. At the molecular level Eg707 overexpression caused an increase in the transcription of SAPK9, a SnRK2 protein kinase family member that is activated by ABA and hyperosmotic stress. Together, the results show that ectopic Eg707 expression influences cell division and differentiation, presumably via altered hormone homeostasis.

THE DEVELOPMENT OF GENETIC RESISTANCE TO PLUM POX VIRUS IN TRANSGENIC NICOTIANA BENTHAMIANA AND PRUNUS DOMESTICA

Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. It is the causal agent of the most devastating viral disease on many stone-fruit spp. The viral disease, known as Sharka in Europe, was recently found in the United States and Canada. Genetic resistance is considered the most effective means to control PPV, but despite over 60 years of extensive screening for germplasm resistant to PPV, few resistant resources have been identified. Here we report engineering genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach. Two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, were cloned into a plant transformation vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo polygalacturonase genomic DNA. These two constructs (pAWp1 and pAWcp) were initially tested in Nicotiana benthamiana. Over 50 transgenic lines transformed with pAWp1, pAWcp and an empty plasmid pAWck (as a control) were generated. The transgenic N. benthamiana plants were mechanically inoculated with PPV. ELISA and RT-PCR analysis revealed that 49.1% of pAWp1-derived lines and 41.2% of pAWcp-derived lines were PPV-negative and 100% of control lines (transformed with pAWck) showed PPV-positive. The pAWp1 plasmid was used to transform plum (Prunus domestica L.). Transgenic plum plants were challenged with PPV by chip-bud inoculation. PPV was detected in all the pAWck-derived plum lines but was not detectable in 50% of pAWp1-derived transgenic lines. Two species of PPV-specific small interfering RNAs, a hallmark of posttranscriptional gene silencing (PTGS), were present in the resistant plum lines, suggesting PTGS is responsible for resistance. The PPV-resistant transgenic plum generated in this study may provide an alternative resistant source for the control of PPV.

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTII fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and eliminating potential promoter‐promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1gl 21 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75mgl 21 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome; 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5yr.

A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants.

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape ( Vitis vinifera)

Activity of three constitutive promoters and enhanced derivatives in transgenic grape ( Vitis vinifera L. cv. Thompson Seedless) was characterized using a bifunctional fusion marker containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes. Relative differences in transient GFP expression and stable transformation efficiencies were used to compare promoter activity. Expression patterns in transformed somatic embryos revealed that the ACT2 promoter from Arabidopsis thaliana, previously shown to be a strong constitutive promoter in A. thaliana and other species, failed to promote strong expression in grape. In contrast, a promoter isolated from cassava vein mosaic virus (CsVMV) supported high levels of transgene expression equivalent to those achieved using an enhanced double cauliflower mosaic virus (CaMV) 35S promoter. Duplication of the 5′-upstream enhancer region of the CsVMV promoter further enhanced its ability to increase transgene expression. However, the pattern of transgene expression driven by these two viral promoters was significantly different at the whole plant level. The enhanced double CaMV 35S promoter was highly active in most tissues and organs including roots, mature leaves, shoot apices and lateral buds. In contrast, the CsVMV promoter and its double enhancer derivative induced relatively weak expression in these tissues. Our results suggest that activity of the CsVMV promoter, in contrast to the CaMV 35S promoter, was under developmental regulation in transgenic grape plants as compared with the CaMV 35S promoter.