Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double‐stranded RNA

Chen, Shuai; Hofius, Daniel; Sonnewald, Uwe; Börnke, Frederik

doi:10.1046/j.1365-313x.2003.01914.x

Cited by 92 publications

(71 citation statements)

References 59 publications

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“…Given the increasing use of the alc system (Ait-ali et al, 2003;Chen et al, 2003;Deveaux et al, 2003;Maizel and Weigel, 2004), there is a need to widen its applicability to plant culture growth conditions. In this article, we report the leaky expression of the alc system in plant tissue cultures and an adaptation of it for use in plant cell suspension cultures, in particular in tobacco BY-2 cells.…”

Section: Discussionmentioning

confidence: 99%

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

et al. 2005

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The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: β-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.

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Section: Discussionmentioning

confidence: 99%

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

et al. 2005

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“…Constructs for ethanol-inducible RNA interference of FLN1 and FLN2 were generated as described (Chen et al, 2003) employing cDNA fragments amplified with specific primers listed in Supplemental Table 1 online. The resulting constructs were transformed into Arabidopsis using the floral dip method (Clough and Bent, 1998).…”

Section: Generation Of Transgenic Arabidopsis Plantsmentioning

confidence: 99%

Plastidial Thioredoxin z Interacts with Two Fructokinase-Like Proteins in a Thiol-Dependent Manner: Evidence for an Essential Role in Chloroplast Development in Arabidopsis and Nicotiana benthamiana

et al. 2010

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Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x-and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEPdependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

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“…Extraction of total RNA from leaf material and northern-blot analysis was performed as described by Chen et al (2003). MP17:GFP specific transcripts were detected using a random-primed [ 32 P]labeled cDNA fragment.…”

Section: Northern-blot Analysismentioning

confidence: 99%

The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein

Kronberg¹,

Vogel²,

Rutten³

et al. 2007

Plant Physiol.

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Ectopic expression of viral movement proteins (MPs) has previously been shown to alter plasmodesmata (PD) function and carbon partitioning in transgenic plants, giving rise to the view of PD being dynamic and highly regulated structures that allow resource allocation to be adapted to environmental and developmental needs. However, most work has been restricted to solanaceous species and the potential use of MP expression to improve biomass and yield parameters has not been addressed in detail. Here we demonstrate that MP-mediated modification of PD function can substantially alter assimilate allocation, biomass production, and reproductive growth in Arabidopsis (Arabidopsis thaliana). These effects were achieved by constitutive expression of the potato leaf roll virus 17-kD MP (MP17) fused to green fluorescent protein (GFP) in different Arabidopsis ecotypes. The resulting transgenic plants were analyzed for PD localization of the MP17:GFP fusion protein and different lines with low to high expression levels were selected for further analysis. Low-level accumulation of MP17 resulted in enhanced sucrose efflux from source leaves and a considerably increased vegetative biomass production. In contrast, high MP17 levels impaired sucrose export, resulting in source leaf-specific carbohydrate accumulation and a strongly reduced vegetative growth. Surprisingly, later during development the MP17-mediated inhibition of resource allocation was reversed, and final seed yield increased in average up to 30% in different transgenic lines as compared to wild-type plants. This resulted in a strongly improved harvest index. The release of the assimilate export block was paralleled by a reduced PD binding of MP17 in senescing leaves, indicating major structural changes of PD during leaf senescence.

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Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double‐stranded RNA

Cited by 92 publications

References 59 publications

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

The alc-GR System. A Modified alc Gene Switch Designed for Use in Plant Tissue Culture

Plastidial Thioredoxin z Interacts with Two Fructokinase-Like Proteins in a Thiol-Dependent Manner: Evidence for an Essential Role in Chloroplast Development in Arabidopsis and Nicotiana benthamiana

The Silver Lining of a Viral Agent: Increasing Seed Yield and Harvest Index in Arabidopsis by Ectopic Expression of the Potato Leaf Roll Virus Movement Protein

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