Improved plant transformation vectors for fluorescent protein tagging

Abstract

Fluorescent protein labelling technologies enable dynamic protein actions to be imaged in living cells and can also be used in conjunction with other methods such as Forster resonance energy transfer and biomolecular fluorescence complementation. In this report, we describe the generation of a series of 23 novel GATEWAY-compatible vectors based on pGreenII and pDH51 backbones with the latest fluorescent protein tags (Cerulean, EGFP and Venus) and the choice of three in planta selection markers. These vectors can be obtained from the Nottingham Arabidopsis Stock Centre (N9819-N9846) and should be a powerful tool box for transgenic research in plants.

Fig. 1

Transient expression of fluorescent proteins in onion epidermal cells. (a) Confocal images of an onion epidermal cell expressing the tomato sub-family II ethylene receptor LeETR4 fused to Cerulean (CFP). (b) Tomato ethylene signalling component LeEIN2-GFP. (c) Venus (YFP) fused to N-terminal ER targeting sequence and C-terminal ER retention signal (HDEL) expressed in an onion cell as the ER control. (d and e) A single onion cell expressing Arabidopsis protein GONST1 fused to Cerulean as a Golgi marker (blue image, d) and GREEN-RIPE fused to Venus (yellow image, e). (f) The Arabidopsis ethylene signalling component RAN1 fused to Cerulean (false colour red) co-expressed with the GREEN-RIPE tagged with Venus (false colour green). (g) Stably transformed Arabidopsis expressing a methyltransferase fused to GFP. (h) Schematic diagrams of the vectors: pDH51-GW-FP (top), pGreenII-35S-GW-FP (middle), pGreenII-MCS-GW-FP (bottom). T, terminator; GW, attR cassette; MCS, multi-cloning site; LB/RB, T-DNA border; marker, in planta selection genes. Scale bars: a 20 μm, b 24 μm, c 22 μm, d 24 μm, e 24 μm, f 16 μm, g 61 μm