Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

Voß, Andreas; Niersbach, Marion; Hain, Rüdiger; Hirsch, Heinz Josef; Liao, Yu Cai; Kreuzaler, Fritz; Fischer, Rainer

doi:10.1007/bf01682088

Cited by 164 publications

(107 citation statements)

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“…On cording to [15], using as templates the plasmids pBTlll and the other hand, pathogen-resistant plants can be obtained by pADI102, containing the cDNA for the light chain and the Fd fragexpressing antibodies against a protein or other organic moment of the MAK33 antibody, respectively [14]. The resulting selecule necessary for pathogenesis [9,10]. Since many interestquence encodes an scFv in the configuration VL-(Gly4Ser)3-VH ( .…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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A gene encoding a single-chain variable (scFv)signal peptide sequence was compared, lower or even no acantibody fragment was expressed as a cytoplasmic and cumulation of the cytosol-targeted protein was observed, both endoplasmic reficulum-targeted protein in transgenic tobacco in leaves [11,12] and in seeds [13]. This suggests a lower staplants. In both cases, the scFv accumulated up to 0.01% of total bility of scFv proteins in the cytoplasm. Yet, intracellular soluble protein (TSP). The same scFv fragment was also expression of an anti-artichoke mottled crinkle virus scFv in produced in the periplasm of Escherichia coll. Measurement of Nicotiana benthamiana generated plants with scFv accumulathe affinity by ELISA indicates that the affinity of the tion levels up to 0.1% of TSP, which resulted in reduced inbacterially made scFv is about 80-fold lower than that of the fection incidence and delayed symptom development [9]. parental Fab fragment. The results suggest that the affinity of the However, few data are available on the efficiency of antigenplant-produced scFv fragments is reduced to a similar extent, binding scFv fragment synthesis and on the affinity of the implying that all the plant-produced scFv fragments are antigen produced scFv fragments for the cognate antigen. binding.To investigate the properties of plant-produced scFv mole- The accumulation levels were determined and the antigenbinding properties of the plant-produced scFv fragments were compared to those of a bacterially produced scFv.

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Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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“…The following plasmid DNA, bacteria, and plants were used in the present study: Plasmid DNA, pTSK (Leech et al, 1998), pUC18 (Messing, 1983, pGEM (Pharmacia, Freiburg, Germany), and pSS (Voss et al, 1995); bacteria, Escherichia coli SCS110 (Stratagene, Heidelberg) and Agrobacterium tumefaciens GV3101 (pMP90RK, Gm R , Km R , and Rif R ) (Koncz and Schell, 1986); plant, Nicotiana tabacum cv Petite Havana SR1. Plants were grown in a greenhouse under a 16-h photoperiod comprising natural daylight.…”

Section: Plasmid Dna Bacteria and Plantsmentioning

confidence: 99%

“…The following targeting cassettes were constructed: T-CHL (chloroplast) was generated by cloning tdc as a NcoI/SalI fragment between the 5Ј cDNA sequence of the potato granule-bound starch synthase chloroplast-targeting signal (CTS; V. Hoppmann, S. Di Fiore, S. Zimmermann, N. Emans, T. Rademacher, R. Fischer, and S. Schillberg, unpublished data) and the 3Ј-c-myc/His6 tags; T-CYT (cytosol) was generated from the constructs scFv24CW (Zimmermann et al, 1998) and scFv24H (Spiegel et al, 1999) by cloning tdc as a NcoI/SalI fragment between the 5Ј ⍀-UTR of the tobacco mosaic virus (Schmitz et al, 1996) and the 3Ј-c-myc/His6 tags; and T-ER (endoplasmic reticulum) was generated from the construct biscFv2429-KDEL (Fischer et al, 1999) by cloning tdc as a NcoI/SalI fragment between the plant codon-optimized 5Ј-light chain leader peptide sequence of the murine antibody 24 (LPL*; Voss et al, 1995) and a 3Ј sequence encoding the KDEL ER retention signal (Munro and Pelham, 1986).…”

Section: Construction Of the Plant Expression Cassettesmentioning

confidence: 99%

“…The recombinant tdc cDNAs including the 5Ј targeting sequences and the 3Ј tags were subcloned as a EcoRI/XbaI fragment into the pSS plant expression vector (Voss et al, 1995) between the constitutive CaMV 35S double enhanced promoter and the CaMV terminator sequence, resulting in the plant expression vectors pT-CHL, pT-CYT, and pT-ER for targeting the TDC protein to the chloroplast, cytosol, and ER of plant cells, respectively (Fig. 1).…”

Section: Construction Of the Plant Expression Cassettesmentioning

confidence: 99%

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Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

Fiore

Leech

et al. 2002

Plant Physiology

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Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of l-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.Plants produce large arrays of chemicals, many of which are referred to as secondary metabolites. Despite the minor role initially assigned to these molecules, secondary metabolites are now considered crucial for the interaction of plants with their environment (Verpoorte, 1998). Many secondary metabolites are also important therapeutic agents or pharmaceuticals, and the generally low abundance to which they accumulate has prompted extensive research into their biosynthetic pathways. To date, few plant secondary metabolic pathways have been fully characterized, and some have been partially characterized, although investigation into many is at an early stage. Nevertheless, it is clear that the biosynthesis of secondary metabolites is under strict developmental, temporal, and spatial control in plants (St. Pierre et al., 1999; De Luca and St. Pierre, 2000). As a consequence of the strictly regulated biosynthesis, natural products accumulate to only trace amounts in plants, and their extraction and purification is often difficult and cost intensive. Attempts to use plant cell cultures as an alternative source to natural products have also been problematic, often due to the lack of fully functional pathways required for the production of the target molecules. With the exceptio...

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“…One approach to protect plants against a viral infection is by the expression of a single chain variable fragment (scFv) antibody directed against that particular virus (Tavladoraki et al, 1993;Voss et al, 1995). This has been demonstrated for the icosahedral Artichoke mottle crinkled tombusvirus (AMCV) and the rod-shaped Tobacco mosaic tobamovirus (TMV).…”

Section: Non-viral Genesmentioning

confidence: 99%

Genetic Engineering of Plants for Resistance to Viruses

Mundembe¹,

Allison²,

Sithole-Niang³

2012

Genetic Engineering - Basics, New Applications and Responsibilities

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Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

Cited by 164 publications

References 30 publications

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

Genetic Engineering of Plants for Resistance to Viruses

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