Inducible Gene Expression Systems for Plants

Borghi, Loris

doi:10.1007/978-1-60761-765-5_5

Cited by 60 publications

(41 citation statements)

References 41 publications

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“…The inducible RAN1 control transgene was constructed using the same pMDC7 vector and expresses a hemagglutinintagged version of the RAN1 (At5g20010) gene. Induction was achieved using the 20 mM estradiol spray from Borghi (2010) or mock induction using the same spray without the inclusion of estradiol. T1 plants for the ind-Athila transgene were sprayed as they transitioned to flowering and then were sprayed every day for 5 d or more before inflorescence tissue collection.…”

Section: Transgene Construction and Expressionmentioning

confidence: 99%

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs

et al. 2013

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Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genomewide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.Transposable elements (TEs) constitute large percentages of both animal and plant genomes. TEs are major targets of multiple endogenous gene-silencing pathways that act to limit their expression and ability to generate new insertions and mutations (for review, see Girard and Hannon, 2008). To study TE silencing, the process has been divided into three distinct mechanisms: the de novo initiation/triggering of silencing, the corrective reestablishment of silencing of TEs that were recently transcriptionally reactivated, and the epigenetic maintenance of TE silencing.In the Arabidopsis (Arabidopsis thaliana) genome, nearly all TEs are found in a transcriptionally silenced state (Lippman et al., 2004). This transcriptional gene silencing is maintained by symmetrical DNA methylation, which is propagated through mitotic cell divisions (for review, see Law and Jacobsen, 2010). In contrast to animals, plants do not erase the DNA methylation patterns of their gametes; therefore, CG and CHG (where H = A, T, or C) symmetrical DNA methylation patterns established in one generation are inherited and act to maintain TE silencing in the next generation through a process termed transgenerational epigenetic inheritance (Mathieu et al., 2007;Becker et al., 2011). In addition to the maintenance of symmetrical methylation, methylation of TEs is continually reinforced through a process of ...

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Section: Transgene Construction and Expressionmentioning

confidence: 99%

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs

et al. 2013

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“…A cell type-specific system provides an additional benefit, as deleterious effects of ectopic gene expression can be avoided. During the past two decades, several different inducible vector systems have been developed (for review, see Moore et al, 2006;Borghi, 2010). Typically, these systems consist of genetic components from heterologous systems to avoid interference with plant signaling networks that are induced by chemical interaction with a transcriptional activator (Moore et al, 2006).…”

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confidence: 99%

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants

et al. 2015

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A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

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“…Conditional expression of transgenes has been approached with various strategies based on promoter sequences of genes responding to either chemicals, such as ethanol, dexamethasone and b-estradiol, or physical stimuli, such as heat shock Borghi, 2010). In this work, we decided to use the binary pER8GW vector (Papdi et al, 2008) derived from the pER8 vector carrying an estrogen receptor-based chemical-inducible system.…”

Section: Discussionmentioning

confidence: 99%

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression

et al. 2014

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SUMMARYTranscription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a b-estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANS-PLANTA lines were tested and confirmed for conditional transgene expression upon b-estradiol treatment. As a proof of concept for the exploitation of this resource, b-estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANS-PLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/ DEAR4 was identified as a regulator of cell death in the hypocotyl-root transition zone. Seeds of TRANS-PLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

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Inducible Gene Expression Systems for Plants

Cited by 60 publications

References 41 publications

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants

The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression

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