Random GFP∷cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency

Abstract

We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3 end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that Ϸ2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP. About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently low information content that they can be generated frequently by chance. The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.

Knocking on the heaven’s wall: pathogenesis of and resistance to biotrophic fungi at the cell wall

“…Arabidopsis transgenic lines that express green fluorescent protein (GFP)::cDNA fusions of plasma-membraneresident proteins are being used as tools in fluorescentimaging studies to visualise the subcellular distribution of various plasma-membrane proteins during the invasive growth of Erysiphe cichoracearum (S Koh, S Somerville, unpublished; [56]). Fluorescent signals were absent from haustorial complexes, suggesting that the extra-haustorial membrane may either lack any protein or contain proteins that are unique to this membrane.…”

The PEN1 Syntaxin Defines a Novel Cellular Compartment upon Fungal Attack and Is Required for the Timely Assembly of Papillae

“…With the plasma membrane marker GFP-LTI6b a slight degree of focal accumulation was detected, but the ratio of the signal at the papillae vs. the signal at the plasma membrane was 3.9-fold less than that seen for GFP-PEN1 (p ϭ 2 e Ϫ6 ). No focal accumulation was detected with a presumptive Golgi marker, with a vacuolar marker (Q5), and with freely soluble cytoplasmic GFP (EGAD; Cutler et al, 2000). A very weak focal accumulation of an ER marker (Q4; Cutler et al, 2004) was sometimes detected, and the ratio of the signal at the attack site vs. the signal at the endomembranes or plasma membrane was at least 5.8-fold less than seen with GFP-PEN1 (p ϭ 0.005).…”

“…This difference in relative intensity for GFP-PEN1 and CFP-SYP122 was highly significant (p ϭ 1.7 e Ϫ6 with the t test; p Ͻ 0.0001 with the nonparametric Mann-Whitney U test). We compared the focal accumulation of GFP-PEN1 with that of two additional GFP-based plasma membrane markers and a number of GFP-based endomembrane markers (described in Cutler et al, 2000). Focal accumulation was not detected with the plasma membrane marker GFP-PIP2a.…”

“…deepgreen.stanford.edu/html/vectors.html; Cutler et al, 2000). Plants were transformed by agrobacterium infiltration as described (Cutler et al, 2000). Western analysis (with the SYP122 antibody, see below) of wild-type plants harboring the CFP-SYP122 fusion detected an additional band c. 30 kDa higher than the endogenous band, showing that the gene fusion was intact.…”

“…The amplicon was digested with EcoRI and BamHI and inserted between the EcoRI and BamHI sites of the ECAD vector, a derivative of EGAD in which GFP was replaced by CFP (see www. deepgreen.stanford.edu/html/vectors.html; Cutler et al, 2000). Plants were transformed by agrobacterium infiltration as described (Cutler et al, 2000).…”