title: Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.
abstract: 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.
authors.affiliation: Biology Department, New York University, New York, New York 10003, USA.
authors.forename: Bastiaan O R
authors.initials: BO
authors.lastname: Bargmann
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Birnbaum
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007210:Indoleacetic Acids / Q000737:chemistry / Q000494:pharmacology; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D009928:Organ Specificity / Q000187:drug effects; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D007210:Indoleacetic Acids; D008164:Luminescent Proteins; D010940:Plant Proteins; D000097573:Red Fluorescent Protein