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BREAKTHROUGH TECHNOLOGIES

Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts^1,[C],[OA]

Center for Genomics and Systems Biology, Biology Department, New York University, New York, New York 10003

Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence-activated cell sorting to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using overexpression. The incorporation of a red fluorescent protein selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or fluorescence-activated cell sorting-mediated isolation and downstream examination of overexpression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the red fluorescent protein/GFP dual labeling capability. In response to manipulation of the auxin response network through overexpression of dominant negative auxin signaling components, we quantify effects on auxin-responsive DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kenneth D. Birnbaum (ken.birnbaum{at}nyu.edu).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.133975

^* Corresponding author; e-mail ken.birnbaum{at}nyu.edu.

Received December 12, 2008; accepted January 20, 2009; published January 23, 2009.