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PubMed1996

Resistance against cucumber mosaic virus in plants expressing the viral replicon.

CMV RNAs 1 and 2 are considered to constitute the viral replicon. Tobacco plants were transformed with either RNA1 or RNA2 to produce plant lines V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 (V2V1) lines that expressed both RNA1 and RNA2. An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. Surprisingly, V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. All the data together suggested that the observed resistance does not fit the criteria for either 'RNA-mediated' or 'replicase-mediated' resistance.

Base Sequence↗

PubMed1992

Expression of a downstream gene from a bicistronic transcription unit in transgenic tobacco plants.

We have constructed a set of plasmids carrying an artificial compact stop-start codon sequence, TGATGTAACATGA, between an upstream open reading frame, terminating at one of the stop codons, and a downstream kanamycin-resistance (KmR)-encoding gene (nptII) initiating at the second ATG. These plasmids were introduced into tobacco protoplasts by direct gene transfer. The efficiency of expression of the downstream nptII gene was measured by scoring the number of KmR transformants. With a closer distance between the functional stop and start codons, a tendency to less efficient expression of nptII was found. The integration and expression of both genes as a bicistronic transcription unit were verified by Southern- and Northern-blot analyses. A possible application of the compact stop-start codon sequence for insertional mutagenesis is discussed.

Base Sequence↗

PubMed1997

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants.

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between -330 to - 200, was shown to be necessary for nitrate induction of beta-glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5' deletions in the -330 to -200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to -230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between -230 to -200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the -300 to -130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the -230 to -181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The -240 to -110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.

Base Sequence↗

PubMed1997

Expression of human muscarinic cholinergic receptors in tobacco.

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0-2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.

Cells, Cultured↗

PubMed2000

Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes.

When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype. These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression. RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype. Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1. This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

Endoribonucleases↗

PubMed1996

Expression of a cyanobacterial delta 6-desaturase gene results in gamma-linolenic acid production in transgenic plants.

Gamma-linolenic acid (GLA), a nutritionally important fatty acid in human and animal diets, is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a fatty acid that could be converted to GLA by the enzyme delta 6-desaturase if it were present. As a first step to producing GLA in oil seed crops, we have cloned a cyanobacterial delta 6-desaturase gene. Expression of this gene in transgenic tobacco resulted in GLA accumulation. Octadecatetraenoic acid, a highly unsaturated, industrially important fatty acid, was also found in transgenic tobacco plants expressing the cyanobacterial delta 6-desaturase. This is the first example of engineering the production of 'novel' polyunsaturated fatty acids in transgenic plants.

Animals↗

PubMed1997

Genetic engineering of parthenocarpic plants.

Transgenic tobacco and eggplants expressing the coding region of the iaaM gene from Pseudomonas syringae pv. savastanoi, under the control of the regulatory sequences of the ovule-specific DefH9 gene from Antirrhinum majus, showed parthenocarpic fruit development. Expression of the DefH9-iaaM chimeric transgene occurs during flower development in both tobacco and eggplant. Seedless fruits were produced by emasculated flowers. When pollinated, the parthenocarpic plants produced fruits containing seeds. In eggplant, the genetic manipulation allowed fruit set and growth under environmental conditions prohibitive for fruit setting in the untransformed line, which did not set fruit at all. Under normal environmental conditions, production of marketable fruits took place from pollinated and unpollinated transgenic flowers, while flowers of untransformed control plants did produce fruits of marketable size only from fertilized flowers.

Chimera↗

PubMed1996

Male sterility in transgenic tobacco plants induced by tapetum-specific deacetylation of the externally applied non-toxic compound N-acetyl-L-phosphinothricin.

A system for the inducible destruction of plant tissues based on the deacetylation of the non-toxic compound N-acetyl-L-phosphinothricin (N-ac-Pt) has been developed. The argE gene product of Escherichia coli, representing a N-acetyl-L-ornithine deacetylase was identified to remove the acetyl-group from N-ac-Pt giving the cytotoxic compound L-phosphinothricin (Pt, glufosinate). Transgenic Nicotiana tabacum plants constitutively expressing the argE gene were constructed. No effect of the bacterial N-acetyl-L-ornithine deacetylase on plant growth and reproduction could be traced. However, application of N-ac-Pt on leaves of the transgenic plants led to the formation of necrotic areas due to the release of Pt. Additionally, due to the uptake of the N-ac-Pt by roots, transgenic shoots grown on medium containing N-ac-Pt bleached within 6-7 days and finally died. Untransformed controls showed no reaction to high amounts of N-ac-Pt applied, either under sterile or under unsterile conditions. In order to construct inducible male-sterile plants, the argE coding region was fused to a DNA fragment carrying sequences homologous to the tobacco TA29 promoter, known to function exclusively in the tapetum. Owing to the tapetum-specific expression of the chimeric gene the application of N-ac-Pt led to empty anthers resulting in male-sterile plants. The sanity of the female reproductive part of the male-sterile flowers could be demonstrated by cross-pollination. Without N-ac-Pt treatment the plants turned out to be completely fertile making fertility restoration in the F1 generation superfluous. The system presented is easy to handle and might be applicable to a wide range of crop plants.

Amidohydrolases↗

PubMed1999

A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences.

A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

Bacterial Proteins↗

PubMed2001

Trans-dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.

Amino Acid Sequence↗

PubMed1991

Gene transfer with subsequent removal of the selection gene from the host genome.

A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host.

Agrobacterium tumefaciens↗

PubMed1993

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.

Base Sequence↗

PubMed1995

Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration.

The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility. We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species. The coding sequence of T-urf13 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Plants expressing high levels of URF13 were methomyl sensitive. Subcellular analysis indicated that URF13 is mainly associated with the mitochondria. Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urf13c construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction. Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria. No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria. There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.

Amino Acid Sequence↗

PubMed1998

Transgenes are dispensable for the RNA degradation step of cosuppression.

Cosuppression results in the degradation of RNA from host genes and homologous transgenes after transcription in the nucleus. By using grafting experiments, we have shown previously that a systemic signal mediates the propagation of cosuppression of Nia host genes and 35S-Nia2 transgenes from silenced 35S-Nia2 transgenic stocks to nonsilenced 35S-Nia2 transgenic scions but not to wild-type scions. Here, we examined the requirements for triggering and maintenance of cosuppression in various types of scions. Grafting-induced silencing occurred in 35S-Nia2 transgenic lines over-accumulating Nia mRNA whether they are able to spontaneously trigger cosuppression or not and in 35S-Nia2 transgene-free plants over-accumulating host Nia mRNA caused by metabolic derepression. When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in the 35S-Nia2 transgene-free plants and in the 35S-Nia2 transgenic lines that are not able to trigger cosuppression spontaneously. Conversely, silencing was maintained in the 35S-Nia2 transgenic lines that are able to trigger cosuppression spontaneously. Our results indicate that the presence of a 35S-Nia2 transgene is dispensable for the RNA degradation step of posttranscriptional silencing when host Nia mRNA over-accumulate above the level of wild-type plants. They also suggest that grafting-induced RNA degradation does not result in the production of the systemic silencing signal required for spontaneous triggering and maintenance.

Genes, Plant↗

PubMed1999

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.

Amitrole↗

PubMedDate not supplied

Effective resistance to potyvirus infection conferred by expression of antisense RNA in transgenic plants.

Approximately 660 nt including the carboxy-terminal portion of the bean yellow mosaic potyvirus (BYMV) coat protein gene, complete 3' noncoding sequence and a short poly(A) tract were introduced to produce antisense RNA in transgenic Nicotiana benthamiana. Original (R0) transformants were selfed, and homozygous second generation (R2) populations challenged with infected sap, purified virus, or viral RNA. One transgenic line of 10 examined was extremely resistant to infection by mechanical inoculation of 100 micrograms/ml BYMV or 50 micrograms/ml BYMV RNA (the highest concentrations tested); no virus could be detected in inoculated leaves of this line. Nine other lines were systemically infected when inoculated with BYMV or BYMV RNA, with initial symptoms indistinguishable from those in nontransgenic plants. One of these lines subsequently developed reduced symptoms and then symptomless, virus-free leaves (complete recovery from BYMV infection), while other lines produced leaves with reduced symptoms and reduced virus titer compared to the controls (partial recovery). No transgenic lines had resistance to infection, nor recovery from symptom expression, following inoculation with pepper mottle or turnip mosaic potyviruses. Antisense RNA from 3' regulatory regions can confer multiple degrees of resistance to potyviruses, including extreme resistanc to infection, presumably by interference in virus replication.

Base Sequence↗

PubMed2000

Mesorhizobium loti increases root-specific expression of a calcium-binding protein homologue identified by promoter tagging in Lotus japonicus.

A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (beta-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the "EF hands" in calmodulin-related proteins, each binding one Ca2+. We have named the gene LjCbp1 (calcium-binding protein). Northern (RNA) analyses showed that this gene is expressed specifically in roots of L. japonicus. Expression was reduced in roots inoculated with non-nodulating M. loti mutants and in progeny homozygous for the T-DNA insertion, suggesting a link between the T-DNA insertion and this gene.

Amino Acid Sequence↗

PubMed1994

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.

Arabidopsis↗