scholarly journals Evaluation of Constitutive Viral Promoters in Transgenic Soybean Roots and Nodules

2008 ◽  
Vol 21 (8) ◽  
pp. 1027-1035 ◽  
Author(s):  
Manjula Govindarajulu ◽  
James M. Elmore ◽  
Thomas Fester ◽  
Christopher G. Taylor
Keyword(s):  
Mosaic Virus ◽  
Hairy Roots ◽  
Target Genes ◽  
Root Nodules ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Symbiotic Interaction ◽  
Strawberry Vein Banding Virus ◽  
Viral Promoters

The efficiency of β-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.

scholarly journals (285) Characterization of a Composite Promoter from Genomic Sequences of Grapevine

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1053C-1053
Author(s):  
Manjul Dutt ◽  
Zhijian T. Li ◽  
Sadanand Dhekney ◽  
Dennis J. Gray
Keyword(s):  
Mosaic Virus ◽  
Genomic Sequence ◽  
Fusion Gene ◽  
Core Promoter ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
2S Albumin ◽  
Wide Range ◽  
Constitutive Gene Expression

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

scholarly journals Nematode-Responsive Activity of the Cauliflower Mosaic Virus 35S Promoter and Its Subdomains

1999 ◽  
Vol 12 (3) ◽  
pp. 189-196 ◽  
Author(s):  
David J. Bertioli ◽  
Matthew Smoker ◽  
Paul R. Burrows
Keyword(s):  
Mosaic Virus ◽  
Giant Cells ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Host Gene Expression ◽  
Root Knot Nematode ◽  
Cyst Nematodes ◽  
Feeding Sites ◽  
Cauliflower Mosaic Virus 35S

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.

Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

1996 ◽  
Vol 26 (6) ◽  
pp. 909-917
Author(s):  
Madoka Gray-Mitsumune ◽  
Bong Y. Yoo ◽  
Pierre J. Charest
Keyword(s):  
Fusion Gene ◽  
Black Spruce ◽  
Gus Activity ◽  
Chimeric Gene ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Zygotic Embryos ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Rbcs Promoter

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

scholarly journals Quantification of the 35S Promoter in DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction and Specificity Assessment on Various Genetically Modified Organisms, Part I: Operating Procedure

2005 ◽  
Vol 88 (2) ◽  
pp. 547-557 ◽  
Author(s):  
Sophie Fernandez ◽  
Chrystèle Charles-Delobel ◽  
Angèle Geldreich ◽  
Georges Berthier ◽  
Francine Boyer ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Collaborative Study ◽  
Cauliflower Mosaic Virus ◽  
Performance Criteria ◽  
35S Promoter ◽  
Conserved Sequence

Abstract A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism® 7700 Sequence Detection System and TaqMan® chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

scholarly journals The Cauliflower Mosaic Virus 35S Promoter Extends into the Transcribed Region

2004 ◽  
Vol 78 (22) ◽  
pp. 12120-12128 ◽  
Author(s):  
Sandra Pauli ◽  
Helen M. Rothnie ◽  
Gang Chen ◽  
Xiaoyuan He ◽  
Thomas Hohn
Keyword(s):  
Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Sequence Motifs ◽  
Transcription Start ◽  
Transcriptional Enhancers ◽  
Enhance Gene Expression ◽  
Enhancer Function ◽  
Nucleotide Region ◽  
Cauliflower Mosaic Virus 35S

ABSTRACT A 60-nucleotide region (S1) downstream of the transcription start site of the cauliflower mosaic virus 35S RNA can enhance gene expression. By using transient expression assays with plant protoplasts, this activity was shown to be at least partially due to the effect of transcriptional enhancers within this region. We identify sequence motifs with enhancer function, which are normally masked by the powerful upstream enhancers of the 35S promoter. A repeated CT-rich motif is involved both in enhancer function and in interaction with plant nuclear proteins. The S1 region can also enhance expression from heterologous promoters.

scholarly journals Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

2004 ◽  
Vol 78 (17) ◽  
pp. 9487-9498 ◽  
Author(s):  
Ramachandran Vanitharani ◽  
Padmanabhan Chellappan ◽  
Justin S. Pita ◽  
Claude M. Fauquet
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
African Cassava Mosaic Virus ◽  
35S Promoter ◽  
Viral Dna ◽  
Posttranscriptional Gene Silencing ◽  
Cassava Mosaic Virus

ABSTRACT Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.

scholarly journals trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants

1999 ◽  
Vol 89 (6) ◽  
pp. 480-486 ◽  
Author(s):  
Stephen F. Hanson ◽  
Douglas P. Maxwell
Keyword(s):  
Dna Replication ◽  
Mosaic Virus ◽  
Severe Disease ◽  
Western Hemisphere ◽  
Cauliflower Mosaic Virus ◽  
Natural Resistance ◽  
Nucleoside Triphosphate ◽  
35S Promoter ◽  
Suspension Cell Culture ◽  
Dna Nicking

Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.

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