Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

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Agrobacterium tumefaciens Transformation and Cotransformation Frequencies of Arabidopsis thaliana Root Explants and Tobacco Protoplasts

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.6.449 ◽

1998 ◽

Vol 11 (6) ◽

pp. 449-457 ◽

Cited By ~ 46

Author(s):

Sylvie De Buck ◽

Anni Jacobs ◽

Marc Van Montagu ◽

Ann Depicker

Keyword(s):

Arabidopsis Thaliana ◽

Agrobacterium Tumefaciens ◽

High Frequency ◽

Dna Marker ◽

Plant Cells ◽

Transformation Frequency ◽

Transformed Cells ◽

Root Explants ◽

Tobacco Protoplasts ◽

Agrobacterium Tumefaciens Transformation

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.

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Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens

Nature ◽

10.1038/318624a0 ◽

1985 ◽

Vol 318 (6047) ◽

pp. 624-629 ◽

Cited By ~ 569

Author(s):

Scott E. Stachel ◽

Eric Messens ◽

Marc Van Montagu ◽

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Dna Transfer ◽

Signal Molecules

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Introduksi Gen DefH9-iaaM dan DefH9-RI-iaaM ke dalam Genom Tanaman Tomat Menggunakan Vektor Agrobacterium tumefaciens

Jurnal AgroBiogen ◽

10.21082/jbio.v6n1.2010.p18-25 ◽

2016 ◽

Vol 6 (1) ◽

pp. 18

Author(s):

Ragapadmi S Purnamaningsih

Keyword(s):

Acetic Acid ◽

Agrobacterium Tumefaciens ◽

Genetic Improvement ◽

Plant Cells ◽

Fruit Production ◽

Total Efficiency ◽

Parthenocarpic Fruit ◽

Tomato Genome ◽

Tomato Cultivars ◽

Indole 3 Acetic Acid

Introduction of DefH9-iaaM and DefH9-RI-iaaM Gene Into Tomato Genome Using Agrobacterium tumefaciens. Ragapadmi Purnamaningsih. Plant genetic improvement can be conducted through genetic engineering. Parthenocarpic fruit production could increase fruit production and its qulities. IAA genes were introduced into three tomato cultivars Ratna, Opal and LV 6117 using two constract genes DefH9-iaaM and DefH9-RI-iaaM. The iaaM gene is able to increase auxin biosynthesis in transgenic plant cells and organs because indol-eacetamide, synthesized by the product of the iaaM gene, is converted either chemically or enzimatically to indole-3-acetic acid (IAA), while the promotor DefH9 enable IAA gene expressed specifically in the ovules. The objectives of this experiment was to identify gene introduction into plant genom of three tomato cultivars. The factors tested were two constract of IAA genes (DefH9-iaaM or DefH9-RI-iaaM), tomato cultivars (Ratna, Opal, and LV 6117) and time of explant inoculation (5, 15, 30 minute). The result showed that the best time inoculation was 5 minute. Otherwise three tomato cultivars response better to DefH9-RI-iaaM than DefH9-iaaM. The total efficiency of regeneration and total efficiency of transformation of both genes were 25.38% and 20.32%. PCR analysis showed that 10 plant have positive PCR, were 1 plant carried (Opal) DefH9-iaaM gene and 9 plant (Ratna, Opal, LV 6117) carried DefH9-RI-iaaM gene.

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Gene-Expression Following T-DNA Transfer Into Plant Cells Is Aphidicolin-Sensitive

Functional Plant Biology ◽

10.1071/pp9940125 ◽

1994 ◽

Vol 21 (2) ◽

pp. 125 ◽

Cited By ~ 1

Author(s):

AM Chaudhury ◽

ES Dennis ◽

RIS Brettell

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Nicotiana Plumbaginifolia ◽

Dna Transfer ◽

Host Cells ◽

35S Promoter ◽

Glucuronidase Activity ◽

Pass Through

A transient assay for gene-expression was used to study the early events of T-DNA transfer. Particularly, it was asked if gene expression following T-DNA transfer required DNA replication in the host cell. A β-glucuronidase gene, linked to a CaMV 35S promoter (35S-GUS, engineered so that it was inactive in Agrobacterium tumefaciens) was introduced into Nicotiana plumbaginifolia protoplasts via a disarmed supervirulent strain of Agrobacterium tumefaciens. High β-glucuronidase activity appeared after 3 days of co-cultivation. The activity required the presence of the vir functions of agrobacteria. The activity was drastically reduced if the plant cells were treated with aphidicolin, an inhibitor of DNA replication in eukaryotic cells. While double-stranded (ds) 35S-GUS DNA, introduced by electroporation, showed undiminished expression in the presence of aphidicolin, gene expression from single-stranded (ss) 35S-GUS DNA was inhibited by aphidicolin. These results suggest that DNA replication in host cells is not required for gene expression if ds-DNA is introduced by electroporation, but is required if ss-DNA is introduced by electroporation, or if DNA is transferred via A. tumefaciens. The findings are consistent with a model of T-DNA transfer in which ss-DNA molecules, once introduced into plant cells, must pass through an aphidicolin sensitive step before they can be transcribed. The simplest interpretation is that the ss-DNA is replicated by the host cell's aphidicolin-sensitive DNA polymerase before being integrated into the host genome.

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Application of sonication-assisted Agrobacterium-mediated transformation in Chenopodium rubrum L

Plant Soil and Environment ◽

10.17221/4122-pse ◽

2011 ◽

Vol 49 (No. 6) ◽

pp. 255-260 ◽

Cited By ~ 17

Author(s):

J.I. Flores Solís ◽

P. Mlejnek ◽

K. Studená ◽

S. Procházka

Keyword(s):

Agrobacterium Tumefaciens ◽

Transient Expression ◽

Chenopodium Rubrum ◽

Limiting Factors ◽

Gus Gene ◽

Efficient Tool ◽

Factors Affecting ◽

Agrobacterium Mediated Transformation ◽

Glucuronidase Activity ◽

Gene Coding

Chenopodium rubrum belongs to the plant species in which standard Agrobacterium-mediated transformation procedures remain inefficient. We demonstrate that the employment of sonication-assisted Agrobacterium-mediated transformation (SAAT) effectively enhanced transient expression of GUS gene coding for b-glucuronidase in Chenopodium rubrum. Further the results indicated that the age of seedlings is one of the limiting factors affecting the potency of Agrobacterium tumefaciens infection. Histochemical detection of b-glucuronidase activity revealed that two-days-old seedlings were much more susceptible to infection than ten-days-old ones. According to our results SAAT technology could provide an efficient tool for obtaining stable transformants when applied to two-days-old seedlings of Chenopodium rubrum.

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Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽

10.1128/mbio.00218-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 33

Author(s):

Julieta Aguilar ◽

Todd A. Cameron ◽

John Zupan ◽

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Secretion System ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Content Type ◽

Protein Substrates ◽

Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

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The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation.

Blood ◽

10.1182/blood.v110.11.1344.1344 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1344-1344 ◽

Cited By ~ 1

Author(s):

Wei Chen ◽

Xueqing Liang ◽

Amanda J. Peterson ◽

David H. Munn ◽

Blazar R. Bruce

Keyword(s):

T Cells ◽

Cell Function ◽

Plasmacytoid Dendritic Cell ◽

Critical Role ◽

Suppressor Cell ◽

Intracellular Enzyme ◽

Cpg Oligodeoxynucleotides ◽

Indoleamine 2,3 Dioxygenase ◽

Tryptophan Degradation ◽

Cpg Oligodeoxynucleotide

Abstract Plasmacytoid dendritic cells (PDCs) are a unique DC subset that plays a critical role in regulating innate and adaptive immune responses. Recently, we have shown that human PDCs activated by CpG oligodeoxynucleotide (CpG ODN) can drive naive, allogeneic CD4+CD25− T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the intracellular mechanism(s) underlying PDC-induced Treg generation is unknown. Here we show human PDCs express high levels of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that catabolizes tryptophan degradation. Triggering of Toll-like receptor 9 (TLR9) with CpG oligodeoxynucleotides activates PDCs to upregulate surface expression of B7 ligands and HLA-DR antigen, significantly increases the expression of IDO, and results in the generation of inducible Tregs from CD4+25− T cells with potent suppressor cell function. Blocking IDO activity with a pharmacologic inhibitor 1-methyl-D-tryptophan (1MT) significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine (KYN), the immediate downstream metabolite of tryptophan, bypasses the 1MT effect, and restores PDC-driven Treg generation. Our results demonstrate that IDO pathway is essential for PDC-driven Treg generation from CD4+25− T cells, and implicates the generation of KYN-pathway metabolites as the critical mediator of this process.

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