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PubMed1997

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato.

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the beta-glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.

Abscisic Acid↗

PubMed1992

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A.

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.

2S Albumins, Plant↗

PubMed1992

Seed-specific repression of GUS activity in tobacco plants by antisense RNA.

beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.

Antigens, Plant↗

PubMed1993

Activity of U-snRNA genes with modified placement of promoter elements in transfected protoplasts and stably transformed tobacco.

In higher plants the promoter elements of pol II- and pol III-transcribed U-snRNA genes are identical, comprising a -30 TATA box and an upstream sequence element, USE. The USE and TATA are centred approximately four and three helical DNA turns apart in pol II and pol III genes, respectively, and it is this difference in the element spacing that determines the RNA polymerase specificity of the gene. In this study we have analyzed the effect of spacing mutations on activity of Arabidopsis U2 and U6 genes in transfected protoplasts of Nicotiana plumbaginifolia and in stably transformed tobacco. In the pol III-transcribed U6 gene the insertions and deletions of either odd or even numbers of half helical turns completely inactivate transcription in transfected protoplasts, consistent with the high conservation of the element spacing found in all plant U-snRNA genes. Surprisingly, while insertions of 50 base pairs (bp) or more into the spacer of the pol II-specific U2 gene inactivate transcription, a deletion of 5 bp or insertions of as much as 20 bp decrease transcription by only 40 to 70%. This relaxed requirement for the conserved element spacing is only seen in transfected protoplasts since the same mutant U2 genes are not transcribed in stably transformed tobacco when transcription takes place from the chromosome. The results provide some clues about possible factor interactions at the promoters of plant U-snRNA genes and also offer an example of major differences in transcription between transiently and stably transformed cells.

Arabidopsis↗

PubMed2001

Edible genetically modified microorganisms and plants for improved health.

The development of new strategies for the delivery of vaccine antigens or immune modulators to the mucosal tissue includes innovative approaches such as the use of genetically modified food microorganisms and plants. Even though the 'proof-of-concept' has recently been established for these two systems, key questions mainly related to efficacy and risk of breaking oral tolerance remain to be critically addressed in the immediate future.

Antigens, Bacterial↗

PubMed2001

Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity.

Cholera toxin B subunit (CTB) mature protein was stably expressed in transgenic tobacco plants under the control of the CaMV 35S promoter and TMV Omega fragment. Fusion of the PR1b signal peptide coding sequence to the CTB mature protein gene increased the expression level by 24-fold. The tobacco-synthesized CTB (tCTB) was purified to homogeneity by a single step of immunoaffinity chromatography. The purified tCTB is predominantly in the form of pentamers with molecular weight identical to the native pentameric CTB, indicating that the PR1b-CTB fusion protein has been properly processed in tobacco cells. Furthermore, by immunodiffusion and immunoelectrophoresis, we have shown that the antigenicity of the purified tCTB is indistinguishable from that of the native CTB protein.

Animals↗

PubMed1995

Light-responsive and transcription-enhancing elements regulate the plastid psbD core promoter.

The psbD operon of higher plant plastids is regulated transcriptionally through the activity of an upstream light-responsive promoter. To identify promoter elements important for the regulation, portions of the tobacco psbD 5' region were fused to the reporter gene, uidA, and were introduced into the tobacco plastid genome by targeted gene insertion. Examination of uidA mRNA accumulation in dark-adapted and light-treated transplastomic plants revealed that a 107 bp segment of psbD 5' sequence was sufficient to promote light-responsive expression of the reporter gene in vivo. The 107 bp promoter region contains three pairs of short, repeated sequences upstream of the core promoter -10/-35 elements. Deletion of the upstream-most A-rich sequences resulted in a 5-fold decrease in reporter gene mRNA accumulation, but did not affect the light response. Additional removal of the second and third repeated elements further reduced the promoter strength approximately 30-fold and almost eliminated the light-dependent accumulation of uidA transcripts. These data indicate that the architecture of chloroplast promoters is more complex than previously assumed, and may comprise general enhancer and regulatory elements in addition to the core promoter motifs. Transcriptional regulation of psbD may be mediated by the chloroplast proteins which were shown to interact with the repeated sequences.

Base Sequence↗

PubMed1995

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.

Cholera Toxin↗

PubMed1994

Beet curly top virus symptom amelioration in Nicotiana benthamiana transformed with a naturally occurring viral subgenomic DNA.

Beet curly top virus (BCTV) infection is associated with the de novo synthesis of a heterogeneous population of subgenomic viral DNAs. Nicotiana benthamiana plants transformed with a partial repeat of one such subgenomic DNA remain susceptible to infection but produce ameliorated symptoms when agroinoculated with BCTV. Transgenic plants contained from 10 to 30% of the amount of viral DNA detected in nontransformed control plants showing severe symptoms. Symptom amelioration is associated with the mobilization of subgenomic DNA from the integrated template and its amplification to approximately one third of the total amount of viral DNA. The amplification in transgenic plants of a specific subgenomic DNA rather than a heterogeneous population implies that mobilization from the integrated template frequently occurs during systemic infection, precluding the accumulation of other subgenomic DNA forms.

DNA, Viral↗

PubMed1996

Resistance to geminivirus infection by virus-induced expression of dianthin in transgenic plants.

Ribosome-inactivating proteins (RIPs) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. Here, the action of dianthin, a potent RIP isolated from Dianthus caryophyllus, has been exploited to engineer resistance to a plant DNA virus, African cassava mosaic virus (ACMV), in transgenic Nicotiana benthamiana. To achieve this, dianthin has been expressed from the ACMV virion-sense promoter that is transactivated by the product of viral gene AC2. This avoids the need for constitutive expression of the RIP, facilitating the regeneration of phenotypically normal plants, and ensures transgene expression is localized to virus-infected cells. When challenged with ACMV, transgenic plants produce atypical necrotic lesions on inoculated leaves, indicative of dianthin expression, viral DNA accumulation is significantly reduced in these tissues, and plants exhibit attenuated systemic symptoms from which they recover. This phenotype holds for isolates of ACMV but not for other geminiviruses, suggesting that AC2 homologues from the latter are unable to efficiently transactivate the ACMV promoter.

Antiviral Agents↗

PubMed1998

Characterization of cucumber mosaic virus. VI. Generation of deletions in defective RNA 3s during passage in transgenic tobacco expressing the 3a gene.

Defective mutants of cucumber mosaic virus (CMV) RNA 3, containing deletions in the 3a gene were passaged in transgenic tobacco plants expressing the CMV 3a gene. After six passages, the various progeny RNA 3 were characterized. In all but one case, the size of the deletion increased. For the NheI-fs RNA 3 mutant of the Fny-strain of CMV (with an 8 nucleotide deletion), the deletion increased in the progeny viral RNA 3 to 570 nucleotides. For a similar frameshift mutant in RNA 3 of the M strain of CMV, either single RNA 3 species (with deletions of 579 or 627 nucleotides) or mixtures of RNA 3 deletion variants were observed in different plants. The DeltaE-H mutant (with a deletion of 202 nucleotides) of Fny-CMV RNA 3 underwent further deletion resulting in the loss of the entire 3a gene and flanking sequences. The DeltaKpnI mutant (deletion of 501 nucleotides) of Fny-CMV RNA 3 underwent a further deletion of 30 nucleotides. Except for the deletion progeny of the DeltaE-H RNA 3 mutant, the other defective RNA 3s all contained inframe deletions. It is proposed that the various deletions were created by different types of recombination and that packaging may be an important factor in the selection of particular defective RNA 3 variants.

Base Sequence↗

PubMed1998

Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes.

Cucumber mosaic cucumovirus (CMV) infection but not tomato black ring nepovirus infection counteracted post-transcriptional gene silencing (PTGS) of nitrate reductase (Nia) or beta-glucuronidase (uidA) transgenes in newly developing leaves of tobacco and Arabidopsis plants. PTGS did not affect meristems of noninfected silenced plants, indicating that the interfering effect of CMV is not likely to occur in the meristem. Models are proposed to explain how CMV (which has no sequence similarity to the Nia or uidA transgenes) can inhibit cellular factors involved in the RNA degradation step of PTGS and/or inhibit the systemic spread of the silencing signal to tissues emerging from the meristem.

Arabidopsis↗

PubMed1992

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme.

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.

Base Sequence↗

PubMed1993

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues.

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene beta-glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.

Acyl Carrier Protein↗

PubMed1995

Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants.

Three constructs were used to study the expression of the avirulence gene Avr9 from the fungal tomato pathogen Cladosporium fulvum in plants. They include pAVIR1, pAVIR2 and pAVIR21, encoding the wild-type AVR9 protein and two hybrid AVR9 proteins containing the signal sequences of the pathogenesis-related proteins PR-S and PR-1a, respectively. Transgenic tobacco plants obtained with the three constructs showed a normal phenotype and produced AVR9 elicitor with the same specific necrosis-inducing activity as the wild-type AVR9 elicitor produced in planta by isolates of C. fulvum containing the Avr9 gene. Level of expression was not correlated with number of T-DNA integrations, but plants homozygous for the Avr9 gene produced more elicitor protein than heterozygous plants. The amino acid sequence of the processed AVR9 peptide present in apoplastic fluid (AF) of pAVIR1 transformed plants producing the wild-type AVR9 elicitor was identical to that of the wild-type AVR9 peptide isolated from C. fulvum-infected tomato leaves. Transgenic Cf0 genotypes of tomato, obtained by transformation with construct pAVIR21, showed a normal phenotype. However, transgenic F1 plants expressing the Avr9 gene, obtained from crossing transgenic Cf0 genotypes with wild-type Cf9 genotypes, showed delayed growth, necrosis and complete plant death indicating that the AVR9 peptide produced in plants carrying the Cf9 gene is deleterious. The necrotic defence response observed in Cf9 genotypes expressing the Avr9 gene support the potential to apply avirulence genes in molecular resistance breeding.

Base Sequence↗

PubMed1995

The expression of an abundant transmitting tract-specific endoglucanase (Sp41) is promoter-dependent and not essential for the reproductive physiology of tobacco.

In angiosperms the interactions between the secretory matrix of the stylar transmitting tract and the growing pollen tubes have central roles in determining a successful fertilization. Sp41 is a major glycosylated component of the soluble proteins of the transmitting tract matrix and exhibits (1-3)-beta-glucanase activity. It is a member of the pathogenesis-related protein superfamily, but shows developmental regulation as opposed to pathogen induction. In order to investigate the mechanisms regulating Sp41 expression, we isolated and characterized genomic clones corresponding to the sp41 alpha gene. Sp41 alpha contains an intervening sequence localized between the sequences encoding for a putative signal peptide and the mature protein. A fragment of 2.5 kb that lies 5' to the coding region of the gene was sufficient to confer transmitting tract specific expression to a beta-glucuronidase reporter gene in transgenic tobacco plants. The sp41 transcripts have unusually long 5'-untranslated sequences. The leader sequences contain small open reading frames, include secondary structures, and may be involved in post-transcriptional regulation. A possible function for Sp41 in reproductive physiology was tested by monitoring tobacco plants transformed with antisense stylar sp41 alpha RNA: Transgenic antisense plants with immunologically and enzymatically undetectable levels of (1-3)-beta-glucanase were obtained and their offspring analyzed. The progeny plants did not show any detectable phenotypic modifications as they had a normal flower morphology and were fully fertile.

Amino Acid Sequence↗

PubMed1995

Transcription of tobacco phytochrome-A genes initiates at multiple start sites and requires multiple cis-acting regulatory elements.

Promoter regions of the Nicotiana tabacum PHYA1 and PHYA2 genes display 89% sequence identity. Analysis of the 5' ends of both the PHYA1 and the PHYA2 transcript revealed multiple, distinct mRNA species, each differing in length and in abundance. The levels of the major PHYA1, PHYA2 transcripts were found to be auto-regulated by phytochrome. This auto-regulation was most efficient in 2-8-day old seedlings. Furthermore, we examined the expression pattern of the PHYA1-GUS reporter gene, containing a 4700 bp PHYA1 promoter fragment or its 5'-deletion derivatives, by GUS histochemistry and by RNase protection assays in transgenic tobacco plants. Our data indicate that the PHYA1 promoter contains three regions which are necessary for the maximum level and regulated expression. We show that a 264 bp promoter fragment contains a cis-regulatory element(s) responsible for expression in the root tips of transgenic seedlings. The major cis-regulatory elements required for high-level transcription and expression in other organs are located in separate regions of the PHYA1 promoter. These data indicate the contribution of multiple cis-regulatory elements for the maximum and regulated expression of tobacco genes coding for phytochrome A protein.

Base Sequence↗

PubMed1995

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model.

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5' ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.

Base Sequence↗