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.

Figure 1.

The pBeaconRFP transient transformation system. A schematic representation of the control vector pMON999_mRFP and pBeaconRFP, a high-copy-number plasmid containing a 35S-driven mRFP positive marker and a Gateway cassette. [See online article for color version of this figure.]

Figure 2.

The ARF-Aux/IAA auxin response pathway. In the absence of auxin, Aux/IAAs repress the activity of ARF transcription factors. Upon perception of auxin, Aux/IAAs are ubiquitinated and degraded in a proteasome-dependent manner. Dominant negative mutant isoforms of Aux/IAAs (e.g. IAA7mII and IAA19mII) can no longer be ubiquitinated and effect a stable repression of ARF function. [See online article for color version of this figure.]

Figure 3.

Repression of DR5∷GFP activation by IAA7mII and IAA19mII. A, A schematic representation of the experiment. Protoplasts derived from the roots of 1-week-old DR5∷GFP Arabidopsis seedlings were transfected with either pMON999_mRFP, expressing only mRFP, or pBeaconRFP, expressing IAA7mII or IAA19mII. After an overnight incubation, protoplast suspensions were treated for 10 h with 5 μm IAA or solvent alone. B, Flow cytometric analysis of transfected and treated protoplast suspensions. The GFP and RFP intensities for individual protoplasts were recorded and are represented in dot plots; 10,000 events are displayed in each plot. Gates were defined to separate blank, GFP-positive, and RFP-positive events. C, A frequency distribution of the GFP signal of events falling within the RFP gate in B. D, Quantification of the mean GFP signal in RFP-positive cells for auxin- and mock-treated protoplasts transformed with the control vector or pBeaconRFP with IAA7mII or IAA19mII. Data are presented in a histogram ± se (n = 798–2,447). E, A 6-h time course of GFP quantification in an independent experiment. Data are presented in a line graph ± se (n = 579–2,390).

Figure 4.

Transcriptional analysis of cell type-specific IAA7mII and IAA19mII expression. A, A schematic representation of the experiment. Protoplasts derived from the roots of 1-week-old P_WER∷GFP seedlings were transfected with either pMON999_mRFP, expressing only mRFP, or pBeaconRFP, expressing IAA7mII or IAA19mII. After an overnight incubation, protoplast suspensions were treated for 3 h with 5 μm IAA or solvent alone. Dual-labeled protoplasts were isolated by FACS and used for microarray analysis. B, Microscopic examination of protoplasts derived from the roots of 1-week-old P_WER∷GFP seedlings that were transfected with pMON999_mRFP. Bar = 50 μm. C, FACS of the transfected protoplast suspensions. Dot plots are shown depicting the controls used to set up the gates: an untransfected protoplast suspension derived from wild-type roots (blank), an untransfected protoplast suspension derived from P_WER∷GFP roots, and a protoplast suspension derived from wild-type roots transfected with pMON999_mRFP. In addition, a dot plot depicting a protoplast suspension derived from P_WER∷GFP roots transfected with pMON999_mRFP is shown; protoplasts falling within the gate marked “double” were sorted and used for microarray analysis. A total of 100,000 events are displayed in each dot plot. D, Transcriptional analysis of sorted protoplasts. A log-scale heat map and a histogram quantifying the differences in gene expression between the various collected protoplasts are shown. The heat map displays all of the genes that exhibit any significant difference between mock and auxin treatment, between transformation with the different vectors, and by interaction level (see “Materials and Methods”) as measured by microarray analysis; rows represent genes and columns represent treatment and transformation vectors (n = 3). The histogram presents the difference in GH3.5 expression (as measured by microarray) between the various collected protoplasts ± sd (n = 3).