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PubMedDate not supplied

Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.

Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene. PMTV CP was expressed in large amounts in some of the primary transformants. The five transgenic lines which produced the most CP were selected for resistance testing. Flowers on transformed plants were allowed to self-fertilize. Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV. Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated. T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated. Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea. In these tests only two plants out of 99 became infected. Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

Base Sequence↗

PubMed1997

Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development.

Pathogenesis-related (PR) proteins form a heterogeneous family of plant proteins that are likely to be involved in defense and are inducible by pathogen attacks. One group of PRs, represented by the subfamily PR-1, are low-molecular-weight proteins of unknown biochemical function. Here we describe the cloning and characterization of two closely related genes encoding a basic and an acidic PR-1 protein (PR1b1 and PR1a2) from tomato (Lycopersicon esculentum). We present a comparative study of the mode of transcriptional regulation of these two genes in transgenic tobacco plants using a series of promoter-GUS fusions. Unexpectedly, the chimeric PR1a2/GUS gene is not induced by pathogenic signals but instead shows constitutive expression with a reproducible developmental expression pattern. It is expressed in shoot meristems, trichomes, and cortical cells as well as in vascular and nearby tissues of the mature stem. This constitutive expression pattern may represent preemption of plant defenses against potential pathogens. Conversely, the chimeric PR1b1/GUS gene does not show any constitutive expression in the plant, but it is transcriptionally activated following pathogen attack. Upon infection by tobacco mosaic virus, the PR1b1 gene is strongly activated locally in tissues undergoing the hypersensitive response but not systemically in uninoculated tissues. Furthermore, its expression is induced by both salicylic acid and ethylene precursors, two signals that coexist and apparently mediate the activation of local defenses during the hypersensitive response. We speculate that the different mode of expression of the two genes presented here, together with that reported previously for the induction of other PR-1 genes in systemic, uninoculated tissues, may all be complementary and necessary for the plant to acquire an efficient refractory state to resist pathogen attacks.

Amino Acid Sequence↗

PubMed1998

Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (beta-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells. However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter. Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems.

DNA, Viral↗

PubMed1993

Stress responses and metabolic regulation of glyceraldehyde-3-phosphate dehydrogenase genes in Arabidopsis.

We report here effects of three environmental conditions, heat shock, anaerobic treatment, and carbon source supply, on expression of nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana. The steady-state mRNA level of the GapC increased when Arabidopsis plants were transferred from normal growth condition to heat-shock, anaerobiosis, or increased sucrose supply conditions. In contrast, the steady-state mRNA levels for GapA and GapB genes were unaffected or decreased transiently under the same treatments. To identify the cis-acting regulatory elements, transgenic tobacco plants containing a 820-bp GapC 5'-flanking DNA fragment and beta-glucuronidase (Gus) fusion were constructed. Analyses of these transgenic plants indicate that this 820-bp DNA fragment is sufficient to confer both heat-shock and anaerobic responses. These results suggest that transcriptional level control is involved in regulation of GapC expression under these stress conditions. Histochemical analysis of Gus activity indicates that expression of the GapC is cell-type specific and is probably linked to the metabolic activity of the cells.

Anaerobiosis↗

PubMed1993

Identification of methyl jasmonate and salicylic acid response elements from the nopaline synthase (nos) promoter.

Transgenic tobacco plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (cat) were studied for their inducibility by salicylic acid (SA) or methyl jasmonate (MJ) treatments. Either chemical significantly increased CAT activity to a level much higher than that achieved by wounding. Northern blot analysis showed a corresponding increase in mRNA levels. After 20 h of induction of flowering plants, the response to MJ treatment was weaker in old leaves compared with young leaves, whereas the SA response was stronger in old leaves. Kinetic experiments showed that the SA response was much faster than the MJ response, suggesting that the induction mechanism of the nos promoter by these chemicals may differ. Deletion analysis showed that both SA and MJ responses require the DNA sequence between -119 and -112 from the transcription initiation site. This region contains the hexamer sequence (TGACGT) that has been found to be an important regulatory element for several promoters. The MJ response was also reduced by deletions of the CAAT box region or the sequence between -112 and -101, whereas the SA response was not significantly affected by these deletions. This suggests that the nos upstream region containing the hexamer motif is essential for the SA or MJ response and that the CAAT box region and the sequence immediately downstream from the hexamer motif are required for maximum induction by MJ.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1994

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

Base Sequence↗

PubMed1994

An auxin-inducible element in soybean SAUR promoters.

The soybean SAUR (Small Auxin-Up RNA) genes are transcriptionally induced by exogenous auxins within a few minutes after hormone application. This response is specifically induced by auxins primarily in epidermal and cortical cells within elongation zones of hypocotyls and epicotyls. We have previously shown that an 832-bp soybean SAUR promoter/beta-glucuronidase (GUS) reporter gene fusion is responsive to auxin in transgenic tobacco plants (Y. Li, G. Hagen, T.J. Guilfoyle [1991] Plant Cell 3: 1167-1175). Similar results were obtained with an 868-bp SAUR 15A promoter-GUS reporter gene in transgenic tobacco (Y. Li, unpublished results). We have now analyzed a soybean SAUR 15A promoter in transgenic tobacco plants using 5' unidirectional deletions, internal deletions and mutations, and gain-of-function assays with a minimal cauliflower mosaic virus 35S promoter. Our results indicate that the distal upstream element/NdeI restriction endonuclease site element (NDE) (B.A. McClure, G. Hagen, C.S. Brown, M.A. Gee, T.J. Guilfoyle [1989] Plant Cell 1: 229-239) in the SAUR 15A promoter is necessary and sufficient for auxin induction. Our results also show that the 30-bp NDE portion of this element is responsible for most, if not all, of the auxin inducibility of the SAUR 15A promoter. The NDE contains two adjacent sequences, TGTCTC and GGTCCCAT, which have been previously identified as putative auxin-responsive elements. We propose that these elements might function independently or together, possibly with an additional element(s), to confer auxin inducibility to the SAUR promoters.

Base Sequence↗

PubMed1994

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.

Antigens, Plant↗

PubMed1995

Phytochrome A overexpression in transgenic tobacco. Correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels.

Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in several plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (GAs) in tobacco (Nicotiana tabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35S promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35S promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ1) promoters. Neither promoter was as effective as CaMV 35S in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome is important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing GA biosynthesis.

Arabidopsis Proteins↗

PubMed1995

The soybean GH2/4 gene that encodes a glutathione S-transferase has a promoter that is activated by a wide range of chemical agents.

Transcriptional activation of the soybean (Glycine max) GH2/4 gene (also referred to as Gmhsp26-A) and increase in abundance of the GH2/4 mRNA (also referred to as pCE54) have been previously shown to occur following treatment of soybean seedlings with auxins, nonauxin analogs, heavy metals, and a variety of other agents. To determine whether the GH2/4 promoter is responsive to an array of different agents, we have analyzed the inducibility of the GH2/4 promoter fused to the beta-glucuronidase reporter gene in transgenic tobacco (Nicotiana tabacum) plants. We have shown that a wide variety of chemical agents induce this promoter in a tissue-specific and concentration-dependent manner. In addition, we have used an affinity-purified antibody raised against recombinant GH2/4 protein to show that the GH2/4 protein increases in response to auxin application and is localized in the cytosol of soybean cells. Recombinant GH2/4 protein can be purified to homogeneity on a glutathione-agarose resin, and the purified protein has glutathione S-transferase activity when assayed with the substrate 1-chloro-2,4-dinitrobenzene.

Base Sequence↗

PubMed1995

Transgenic analysis of a hybrid poplar wound-inducible promoter reveals developmental patterns of expression similar to that of storage protein genes.

The wound-inducible win3 multigene family from hybrid poplars (Populus trichocarpa x Populus deltoides) encodes proteins with structural similarities with Kunitz-type protease inhibitors (H.D. Bradshaw Jr., J.B. Hollick, T.J. Parsons, H.R.G. Clarke, M.P. Gordon [1990] Plant Mol Biol 14: 51-59), and at least one member, win3.12, is transcribed de novo in the injured and uninjured leaves of wounded trees (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). A previous study demonstrated that 1352 bp of 5' flanking DNA from the win3.12 gene confers local wound-regulated expression of the beta-glucuronidase gene in transgenic tobacco (Nicotiana tabacum cv Xanthi n.c.) (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). We extend this transgenic analysis here by examining the developmental regulation and systemic wound induction conferred by the same transgene construct in tobacco. Biochemical and histochemical surveys of beta-glucuronidase activity are described for four, independent transgenic lines. The observed spatial and temporal expression patterns coincide with dormant storage tissues and with previously described expression patterns for both seed and vegetative storage protein genes. Developmental northern blot analysis of win3 RNA levels in poplar seeds confirms that proper temporal expression of the reporter gene is maintained during tobacco seed maturation. These results demonstrate that a putative Kunitz-type protease inhibitor can be wound inducible in addition to being expressed in developing seeds.

Gene Expression Regulation, Developmental↗

PubMed1995

Induction of nopaline synthase promoter activity by H2O2 has no direct correlation with salicylic acid.

Transgenic tobacco (Nicotiana tabacum L.) plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (caf) were tested for their response to treatment with H2O2. The nos promoter-driven CAT activity increased significantly by addition of H2O2, reaching the maximum level at 15 mM. Kinetic analysis for CAT activity showed that induction by H2O2 was similar to that of methyl jasmonate (MJ), but was much slower than induction by salicylic acid (SA). Time-course experiments for mRNA level also revealed that the response to H2O2 treatment was similar to that of MJ. The nos promoter displayed a rapid and transient induction of mRNA with SA treatment, with the maximum levels occurring at 3 h, whereas the levels induced by H2O2 or MJ treatment increased continuously during the 11-h experimental period. The antioxidants N-acetyl-L-cysteine and catechol did not alter the SA effect. The responses of the nos promoter to H2O2, MJ, and wounding were significantly reduced by deletions of the CAAT box region and the sequence between -112 and -101. However, these deletions did not significantly alter the SA response. This suggests that H2O2 may have a different mechanism from that of SA for inducing nos promotor activity.

Acetylcysteine↗

PubMed1997

Characterization and functional expression of a ubiquitously expressed tomato pectin methylesterase.

Pectin methylesterase (PME), a ubiquitous enzyme in plants, de-esterifies the methoxylated pectin in the plant cell wall. We have characterized a PME gene (designated as pmeu1) from tomato (Lycopersicon esculentum) with an expression that is higher in younger root, leaf, and fruit tissues than in older tissues. Hypocotyls and epicotyls show higher accumulation of pmeu1 transcripts compared with cotyledons. pmeu1 represents a single-copy gene in the tomato genome. Comparison of the deduced amino acid sequence of pmeu1 with other PME homologs showed that the N-terminal halves are highly variable, and the C-terminal halves are relatively conserved in plant PMEs. Constitutive expression of a fruit-specific PME antisense gene does not affect the level of pmeu1 transcripts in vegetative tissues but does lower the level of PMEU1 mRNA in developing tomato fruits. These results suggest that there exists developmentally regulated silencing of pmeu1 by a heterologous PME antisense gene. Expression of pmeu1 in tobacco (Nicotiana tabacum) under the control of the cauliflower mosaic virus 35S promoter caused up to a 4-fold increase in PME specific activity that was correlated with the accumulation of PMEU1 mRNA. In vitro transcription-translation analyses show that pmeu1 encodes a 64-kD polypeptide, whereas transgenic tobacco plants expressing pmeu1 accumulate a new 37-kD polypeptide, suggesting extensive posttranslational processing of PMEU1. These results are the first evidence, to our knowledge, of the functional characterization of a PME gene and the extensive modification of the encoded polypeptide.

Amino Acid Sequence↗

PubMed1997

Promoter analysis and expression of a phospholipase D gene from castor bean.

The expression of a castor bean (Ricinus communis L.) phospholipase D (PLD; EC 3.1.4.4) gene has been studied by examining its promoter activity in transgenic tobacco (Nicotiana tabacum) carrying a PLD promoter-glucuronidase transgene and by monitoring the levels of PLD mRNA in castor bean. Sequence and the 5' truncation analyses revealed that the 5' flanking region from nucleotide -1200 to -730 is required for the regulation and basal function of the PLD promoter. The PLD promoter in vegetative tissues is highly active in the rapidly growing regions such as the shoot apex and the secondary meristem producing axillary buds and vascular tissues of young leaves and stems. The PLD promoter activity in floral tissues was high in stigma, ovary, and pollen grains, but low in petals, sepals, the epidermis of anthers, styles, and filaments. The PLD promoter activity was enhanced by abscisic acid. Northern-blot analysis of PLD in castor bean showed that the PLD mRNA levels were high in young and metabolically more active tissues such as expanding leaves, hypocotyl hooks, developing seeds, and young seedlings, and they decreased in mature tissues such as fully expanded leaves and developed seeds. These patterns of expression suggest a role of PLD in rapid cell growth, proliferation, and reproduction.

Base Sequence↗

PubMed1999

An imperfect heat shock element and different upstream sequences are required for the seed-specific expression of a small heat shock protein gene.

Chimeric constructs containing the promoter and upstream sequences of Ha hsp17.6 G1, a small heat shock protein gene, reproduced in transgenic tobacco (Nicotiana tabacum) its unique seed-specific expression patterns previously reported in sunflower. These constructs did not respond to heat shock, but were expressed without exogenous stress during late zygotic embryogenesis coincident with seed desiccation. Site-directed mutagenesis of its distal and imperfect heat shock element strongly impaired in vitro heat shock transcription factor binding and transgene expression in seeds. Deletion analyses of upstream sequences indicated the contribution of additional cis-acting elements with either positive or negative effects on transgene expression. These results show differences in the transcriptional activation through the heat shock element of small heat shock protein gene promoters in seeds compared with the heat shock response. In addition, they suggest that heat shock transcription factors and other distinct trans-acting factors cooperate in the regulation of Ha hsp17.6 G1 during seed desiccation.

Gene Expression Regulation, Plant↗

PubMed1999

Wound-induced expression of the FAD7 gene is mediated by different regulatory domains of its promoter in leaves/stems and roots.

The FAD7 gene is expressed preferentially in the chlorophyllous tissues of unwounded plants. Wounding activates the expression of the FAD7 gene not only in chlorophyllous tissues, but also in nonchlorophyllous tissues of stems and roots. Our previous study suggested that wound-responsive transcriptional activation by the FAD7 promoter in leaves/stems and roots is brought about by a jasmonic acid (JA)-independent and JA-dependent signaling pathway, respectively. In this paper, we show that a specific region (from -259 to -198) in the FAD7 promoter is required for wound-activated expression of this gene in leaves and stems, while another region (from -521 to -363) is necessary not only for wound-activated but also for JA-responsive expression of this gene in roots. Thus, different regulatory regions of the FAD7 promoter mediate distinct wound-induced expression of this gene in leaves/stems and roots. Gel mobility shift assays revealed the wound-inducible DNA-binding activity to the -242/-223 region in both stem and leaf nuclear extracts. In fact, deletion of this region abolished wound response of the FAD7 promoter, suggesting the in vivo role of this site. Furthermore, we detected root nuclear factors interacting with the region from -433 to -363 of this promoter. Wounding and methyl jasmonate treatments induced differently these DNA-binding activities. These results suggest that different regulatory mechanisms mediate the wound-induced expression of the FAD7 gene in aerial and subterranean organs.

Acetates↗

PubMed2001

Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants.

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.

Actinomyces↗

PubMed1989

cis-analysis of the wound-inducible promoter wun1 in transgenic tobacco plants and histochemical localization of its expression.

The 5' region of the wound-inducible gene wun1, derived from potato, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5' deletion fragments were linked to the reporter gene beta-glucuronidase (GUS) as transcriptional fusions, and the expression of these chimeric genes was analyzed in leaf tissue. Sequences 111 base pairs upstream of the transcriptional start site were not able to drive the GUS expression over background levels, whereas sequences between -111 and -571 showed a slightly higher activity with equal levels of transcription in wounded and nonwounded tissue. The addition of further upstream sequences (-571 to -1022) enhanced the level of expression by a factor between 13 and 370. The expression driven by this fragment was inducible by a factor of twofold to ninefold by wounding. Histochemical analysis of different tissue from transgenic plants that contain wun1-GUS fusions demonstrates wound-inducible and cell-specific wun1 promoter activity in plants containing the -1022-base pair fragment. The location of GUS activity appears to be cell-specific, being highest in epidermal cells of leaves and stems and lower in vascular cells. Activity was reduced to levels that could not be detected by histochemical staining in leaves, stems, and roots of plants containing the deleted promoter fragments. Plants that contain the different deletion constructs and plants that carry the -1022-base pair fragment show high expression in anthers and pollen grains that could not be stimulated by wounding.

Base Sequence↗