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PubMed1989

Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. We have constructed Nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, AL1, AL2, and AL3, from the left side of TGMV A. In the transgenic plants, the AL open reading frames are under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In TGMV infectivity assays, seven of 10 transgenic lines complemented TGMV A variants with mutations in AL1, AL2, or AL3 when co-inoculated with the B component. The 35S-AL construct was transcribed as a single RNA species in the transgenic plants, indicating that AL1, AL2, and AL3 were expressed from a polycistronic mRNA. This differs from the complex transcription pattern in TGMV-infected plants, which contains five AL transcripts. There was no quantitative correlation between the efficiency of complementation in the infectivity assay and the level of expression of transgenic AL RNA in the leaves of a transgenic line. One line that failed to complement defects in AL1, AL2, and AL3 in infectivity assays contained high levels of transgenic AL RNA and functional AL1 protein. These results provide evidence that chromosomal position can affect the cell- and tissue-specific transcription of the 35S promoter in transgenic plants. Comparison of the complementing plants and wild-type infected plants may provide insight into the TGMV infection process and the use of the CaMV 35S promoter for gene expression in transgenic plants.

Base Sequence↗
PubMed1996

Bean ribonuclease-like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression.

The intracellular pathogenesis-related (PR) proteins of common bean (Phaseolus vulgaris L.) are encoded by a highly polymorphic family of at least 20 genes. One member, the Ypr10*c gene, has been isolated and characterised. The deduced amino acid sequence of the encoded protein, PR-10, exhibits similarities to tree-pollen allergens, to food allergens from celery and apple and to ginseng ribonuclease peptide sequences. We show by RNA blot analysis that the Ypr10 gene family, including Ypr10*c, is strongly expressed in bean roots. In leaves Ypr10 transcript levels are low in young and mature stages but are elevated during senescence and in diseased states. Dark treatment of leaves causes strong induction of Ypr10 transcripts, which is reversible by light, and diurnal rhythms of transcript accumulation during the night are observed. Ypr10 genes are responsive to external stimuli related to pathogen-defence such as glutathione or salicylic acid. Transcriptional activity of a Ypr10*c promoter-beta-glucuronidase fusion gene in transgenic tobacco was observed in roots, in developing xylem and phloem of stems, and in the blade of senescent leaves, with highest levels at the onset of senescence. The most striking characteristic of developmental expression was the specific localisation of beta-glucuronidase activity in the transmitting tract of styles in flowers at anthesis. Feeding of various pathogen-related and stress-related stimuli to young tobacco leaves led to accumulation of GUS activity in leaf blades. We identify considerable spatio-temporal similarities between reported expression patterns of Ypr10 genes and ribonuclease genes, which, together with the significant sequence similarity to the ginseng ribonuclease, support the hypothesis of a ribonuclease function for PR-10 proteins and allow the prediction of possible biological roles.

Allergens↗
PubMed2026

Development of a highly efficient prime editing platform for cucurbits enables breeding of multi-disease-resistant cucumber.

The prime editing (PE) system is a precise genome editing technology that works efficiently in monocots; however, its application is limited by low editing efficiency in dicots, particularly Cucurbitaceae and Solanaceae plants. Here, we first significantly improved the transformation efficiency by introducing spectinomycin in cucurbits, then used the tomato elongation factor 1-alpha (SlEF1α) promoter to enhance PE protein expression, and incorporated the Csy4 ribonuclease to process pegRNAs, collectively addressing multiple constraints limiting PE efficiency in cucurbits. The optimized PE systems, particularly Csy4-PE6d, achieved an average desired editing frequency of 80.83% at targeted loci in cucumber via stable genetic transformation, with frequencies reaching up to 100% at certain sites. Moreover, Csy4-PE6d generated homozygous edits in 36.43% of transgenic lines and demonstrated robust editing activity in melon, pumpkin, and potato. Using the Csy4-PE6d tool, we generated heritable edited cucumber lines with dual resistance to bacterial angular leaf spot and downy mildew by targeting the CsSGR gene. Collectively, this optimized system substantially enhances PE efficiency in Cucurbit crops, providing an effective solution to common challenges such as low editing efficiency and limited heritability in these species.

Disease Resistance↗
PubMed1995

A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Amino Acid Sequence↗
PubMed1997

Abortive gap repair: underlying mechanism for Ds element formation.

The mechanism by which the maize autonomous Ac transposable element gives rise to nonautonomous Ds elements is largely unknown. Sequence analysis of native maize Ds elements indicates a complex chimeric structure formed through deletions of Ac sequences with or without insertions of Ac-unrelated sequence blocks. These blocks are often flanked by short stretches of reshuffled and duplicated Ac sequences. To better understand the mechanism leading to Ds formation, we designed an assay for detecting alterations in Ac using transgenic tobacco plants carrying a single copy of Ac. We found frequent de novo alterations in Ac which were excision rather than sequence dependent, occurring within Ac but not within an almost identical Ds element and not within a stable transposase-producing gene. The de novo DNA rearrangements consisted of internal deletions with breakpoints usually occurring at short repeats and, in some cases, of duplication of Ac sequences or insertion of Ac-unrelated fragments. The ancient maize Ds elements and the young Ds elements in transgenic tobacco showed similar rearrangements, suggesting that Ac-Ds elements evolve rapidly, more so than stable genes, through deletions, duplications, and reshuffling of their own sequences and through capturing of unrelated sequences. The data presented here suggest that abortive Ac-induced gap repair, through the synthesis-dependent strand-annealing pathway, is the underlying mechanism for Ds element formation.

DNA Repair↗
PubMed2001

Transformation of Lotus japonicus using the herbicide resistance bar gene as a selectable marker.

Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.

Acetyltransferases↗
PubMed2000

[Genetic transformation of autotetraploid Isatis indigotica fort. induced by Ri T-DNA and plant regeneration].

OBJECTIVE: To establish an effective system for genetic transformation of autotetraploid Isatis indigotica by Agrobacterium rhizogenes. METHOD: Agrobacterium rhizogenes strains R1601, ATCC15834 and A4 were employed to induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots were developed into regenerated plants on solid MS media with different Kinds of plant growth regulators. RESULT: Root induction differed obviously. By three Agrobacterium strains. Hairy roots grew rapidly on solid MS medium without plant growth regulators and showed the typical hairy root phenotype: profuse branching, high density of root hairs and plagiotropism. And the transformation of Ri T-DNA was confirmed by opine analysis. The biomass of hairy roots increased nearly 35 times regulator-free MS after suspended in plant growth two weeks. On solid MS medium with BA, adventitious buds were differentiated directly from hairy roots without callus formation. All of the adventitious buds were rooted on root induction medium and developed into regenerated plants. Opine analysis indicated the integration of Ri T-DNA in the transformed plants. CONCLUSION: Agrobacterium rhizogene can induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots can develop into regenerated plants.

Coculture Techniques↗
PubMed2006

[Transformation of embryogenic Calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) mediated by agrobacterium].

Formation of embryogenic calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) was induced from mature seeds as explants, and proliferated on MS medium containing 2,4-D 5.0 mg/L and KT 0.05 mg/L. An effective and stable callus regeneration system was established by optimizing the culture conditions (Tables 1, 2 and Fig.2). After the calli were subcultured 8 weeks, selected the whitish-yellow-coloured compact nodular calli that transformed with plasmid pCAMBIA1304 carrying hygromycin resistance gene (hptII) and Pseudomonas pseudoalcaligenes insecticidal protein gene (ppIP), which was mediated by an Agrobacterium strain EHA105. Resistant plants were obtained after hygromycin selection (Figs.3, 4). Some important factors that affect the transformation efficiency were studied, which included selection pressure, time of embryogenic calli proliferation, OD value of Agrobacterium suspension, temperature, medium and time of co-cultivation, and concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformation plant regeneration. This research is the first successful genetic transformation of Elymus sibiricus L. cv. Chuancao No.2 mediated by Agrobacterium.

Culture Media↗
PubMed1994

Integration and expression of human growth hormone gene in Caladium bicolor.

Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9. It has been introduced into the agrobacterium containing plasmid pGV3850. The recombinant Ti plasmid pGL198(hGH) has been obtained by homologous recombination. The monocotyledon Caladium bicolor has been transferred with pGL198 (hGH) with the leaf-disk co-cultivation method, and transgenic plants have been regenerated. The results of nopaline analysis, NPT II detection Southern blot and Western blot show that the hGH gene was integrated into the genome of Caladium bicolor, and a 22-kD protein was synthesized in the transgenic plants.

Gene Transfer Techniques↗
PubMed1997

Characterization of a hexamer motif and b element of the nopaline synthase (nos) promoter.

Nopaline synthase (nos) is of bacterial origin and expressed in plant tissues to generate an unusual compound, nopaline, which is secreted into the environment where Agrobacterium tumefaciens uses it as a nutrient source. The nos promoter contains three distinctive regions which are important for the promoter activity. Among these, the upstream region between -131 and -112 is essential for the nos promoter. The upstream region consists of two hexamer motifs which are separated by an eight nucleotide sequence. In this study, we have investigated the role of the hexamer motif that is located between -117 and -112. Increasing the distance between the hexamer and the downstream regulatory region significantly reduced the promoter activity, indicating that a proper distance between the regulatory elements is needed for an optimum level of activity. Insertion of a single copy of the hexamer enhanced the promoter in both orientations. Point mutations in the inserted hexamer sequence reduced the enhancing effect. These results confirmed that the hexamer motif is important for the nos promoter activity. The b sequence that is located immediately downstream of the hexamer motif did not function by itself, but together with the hexamer it appeared to enhance the promoter activity. These indicate that the sequence surrounding of the upstream regulatory element is also involved in controlling the promoter function.

Agrobacterium tumefaciens↗
PubMed1997

[Transfer of bacterial genes for proline synthesis in plants and their expression by various plant promotors].

Accumulation of free proline (Pro) by bacteria and plants serves as a mechanism protecting these organisms from abiotic stress. We introduced two E. coli genes, proBosm, encoding a mutant variant of the first enzyme of the Pro biosynthetic pathway, gamma-glutamyl kinase, and proA, encoding the second enzyme of the Pro pathway, glutamyl-gamma-semialdehyde dehydrogenase, into tobacco plants. The spontaneous proBosm mutation was selected for Pro overproduction and mapped to the N-terminus of a polypeptide chain; it was found to result from one amino-acid substitution. Two E. coli genes were simultaneously introduced into tobacco plants, each under the control of a strong constitutive CaMV 35S promoter, which contained a duplicated sequence of enhancer, or the root-specific Pmas promoter sequence. Transgenic plants were characterized by Pro overproduction, increased resistance to L-azetidine-2-carboxylic acid (a toxic analog of Pro), and tolerance to salt stress.

Agrobacterium tumefaciens↗
PubMed1996

Modified 2S albumins with improved tryptophan content are correctly expressed in transgenic tobacco plants.

Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the protein's nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.

Agrobacterium tumefaciens↗
PubMed2001

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Fabaceae↗
PubMed2006

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.

Biotechnology↗
PubMed1994

Endoplasmic reticulum targeting of active modified beta-glucuronidase (GUS) in transgenic tobacco plants.

We have investigated targeting to the endoplasmic reticulum (ER) of wild-type GUS and a modified form (GUS S358) by making an N-terminal fusion of the beta-glucuronidase (GUS) enzyme with the wheat alpha-amylase signal peptide. In vitro studies demonstrated that the modified GUS (S358) lacked the glycosylation site present within the wild-type enzyme. Analysis of transgenic tobacco plants revealed that the modified GUS enzyme retained activity upon passage to the ER. When further experiments were carried out to determine the cellular location of the modified GUS enzyme, it was found that (contrary to expectation) the majority of GUS activity was retained within the cell and was not secreted to the cell surface via the default pathway. The data indicated that the modified GUS enzyme is an unsuitable reporter enzyme for studying protein secretion.

Amino Acid Sequence↗
PubMed1997

Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA.

Tobacco plants were transformed with constructs in which the transgene was a cDNA of replicating potato virus X (PVX) RNA. The constructs, referred to here as amplicons, were the intact genome of PVX and PVX constructs modified to carry the beta-glucuronidase (GUS) reporter gene either as an additional gene or as a replacement for the coat protein gene (PVX/GUS/CP and PVX/GUS respectively). Transformed plants carrying these constructs displayed several phenotypes that we attribute to post-transcriptional gene silencing. These phenotypes include the absence of viral symptoms, low accumulation of transgene-derived RNA, extreme strain-specific resistance against PVX, low and non-uniform GUS expression (in the PVX/GUS and PVX/GUS/CP plants) and suppression of transiently expressed RNA sharing homology with the transgene. Importantly, the amplicon-mediated gene silencing was exhibited in all lines tested. There was no evidence of gene silencing in seven lines expressing a PVX RNA that was unable to replicate. From these data we conclude that the replicating viral RNA is a potent trigger of gene silencing. Moreover, amplicon-mediated gene silencing provides an important new strategy for the consistent activation of gene silencing in transgenic plants.

Cloning, Molecular↗