Transient Transactivation Studies in Nicotiana benthamiana Leaves

Transitory gene expression systems in Nicotiana benthamiana leaves, in combination with the use of gene silencing suppressors as the p19 or HC-pro proteins that allow for elevated levels of gene expression, have proven to be a highly versatile tool to analyze transcriptional function of DNA binding factors in the activated or repressed expression of their gene targets. This experimental setup uses Agrobacterium-mediated infection to deliver the various DNA constructs into the cell, and offers the advantage with respect to mesophyll protoplast transfection procedures that it entails a much easier protocol, in addition to preserving the intact leaf tissue, thus being more amenable to the study of wound and stress signaling pathways or to the functional analyses of regulators that respond to Ca⁺² signatures. Furthermore, by using reporter constructs based on the LUCIFERASE (LUC) gene, which does not require a destructive determination assay, this expression system can be used to test for changes in gene activity over time or in response to various treatments, thus providing a comprehensive understanding of the signaling pathways that modulate activity of the expressed regulators and therefore their in vivo function in the control of the analyzed promoter.