Predictable activation of tissue‐specific expression from a single gene locus using the pOp/LhG4 transactivation system in Arabidopsis

Abstract

SummaryThe pOp / LhG4 transcription factor system was used to determine whether the synthetic pOp promoter, integrated at one position in the Arabidopsis genome, could be efficiently and faithfully activated by the heterologous transcription factor, LhG4, expressed in a variety of different patterns. This is a precondition for the development and exploitation of large collections of LhG4 activation lines that direct predictable tissue-specific expression of transgenes. We selected a pOp-GUS reporter insertion that was efficiently activated after crossing to an activator line that expressed the synthetic transcription factor LhG4 from the Cauliflower Mosaic Virus 35S promoter. This reporter line, pOp-GUS(g2), was then combined with activator loci that expressed LhG4 from one of seven different promoters, each with a different tissue specificity. pOp-GUS(g2) was activated faithfully in combination with six of these seven activator constructs, but generated an unexpected expression pattern in combination with the seventh construct, a fusion to a cyclin promoter (CYC-LhG4). The aberrant expression pattern could be attributed to the pOp-GUS(g2) insertion site, as the CYC-LhG4 activator lines directed the expected pattern of expression from a second pOp-GUS insertion. These results show that it is feasible to construct an activator collection in which LhG4 is expressed from diverse promoters or enhancer traps, but that individual pOp reporter loci can vary in their competence to respond to certain activator patterns. We discuss the implications for the design and use of mis-expression technology in Arabidopsis .

A Versatile and Reliable Two-Component System for Tissue-Specific Gene Induction in Arabidopsis

“…These results demonstrate that the system can be used to generate a library of tissuespecific transactivator plant lines for the activation of LexA responder T-DNAs and that the genomic location of the activator or responder is equally likely to affect the reliability of the system. Such efficiency could not be easily achieved using systems in which gene expression is more sensitive to the position of the responder T-DNA (Baroux et al, 2005). In the system described by Baroux et al (2005), a similar percentage of plant lines (to data presented here) showed transactivated GUS expression (i.e.…”

“…Such efficiency could not be easily achieved using systems in which gene expression is more sensitive to the position of the responder T-DNA (Baroux et al, 2005). In the system described by Baroux et al (2005), a similar percentage of plant lines (to data presented here) showed transactivated GUS expression (i.e. 86% of plant lines with GUS responders inserted at 37 independent loci), but expression was often weak (e.g.…”

“…These features make it difficult to produce distinct sectors of induced and uninduced gene expression essential to determine the cell autonomy of a phenotype and to address hitherto intractable problems encountered during reproductive developmental studies (where gene activation can prevent production of viable seeds). Some systems, based on nonvolatile inducers, have shown leaky activity (Martinez et al, 1999;De Veylder et al, 2000) or an inability to reliably activate responder T-DNAs randomly inserted in the genome (Baroux et al, 2005). This prohibits their use in precise activation-tagging screens.…”

Using transgenic modulation of protein synthesis and accumulation to probe protein signaling networks in Arabidopsis thaliana

“…5 In particular, the mis-expression of genes that cause lethality or strong pleiotropic effects cannot be accomplished through activation tagging. 6 Another concern of activation tagging is whether the gain-of-function phenotype of a mutant reflects the normal biological role of a gene. Notably, the CaMV 35S enhancers can lead to enhancement of endogenous gene expression and the resultant phenotype, as opposed to ectopic overexpression or mis-expression driven by native promoters, which would be more likely to reflect the normal role of the activated gene.…”

“…These technologies can be implemented easily for a single gene using specific constructs that direct its down or upregulation under constitutive, tissue-specific or inducible conditions. 6 Developmental processes and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Most systems that manipulate gene expression allow control in one dimension, space or time.…”