Introduction

Recently, a class of fluoresceinrelated protein labeling reagents was developed by Griffin et al. (1) based on the interaction between FlAsHs-EDT₂ (a nonfluorescent biarsenical derivative of fluorescein, bis-ethanedithiol adduct) and a small motif containing the sequence Cys-Cys-Xaa-Xaa-Cys-Cys. FlAsH-EDT₂ and ReAsH-EDT₂, a derivative of the red fluorophore resorufin (Figure 1A), bind with high affinity and specificity to the tetracysteine (TC) motif, becoming the complex TC-(Re)FlAsH, which is strongly fluorescent. This binding is based on the four covalent bonds between the two arsenic groups of the FlAsH with the two thiol groups in the TC-tag. The tightest binding is found when the TC-tag contains Pro-Gly between the cysteine pairs (2). The major advantages of the TC over other technologies for fluorescent protein labeling are the relatively small size of the tag and the ability to pulse-chase the fluorophore (2).

Figure 1. Structures of synthetic glycomodule peptides (SynGMs) and FlAsH/ReAsH dyes. (Click to enlarge)

FlAsH-TC labeling was used to study several in vivo cellular events and subcellular structures in animal cells, such as β-tubulin dynamics (3), Ebola virus matrix protein (4), conformational changes in a G protein-coupled receptor (5), and protein misfolding in vivo (6). Also, correlation was achieved between optical and electron microscopy (EM)-localization for connexin 43 using the same tag (7). Similar methodology has been employed in Escherichia coli (6), mammalian cells (2,7), and yeast (8), but use in plant cells has not been previously described.

In the present work, the expression of several synthetic glycomodule peptides (SynGMs) containing the arabinogalactan protein (AGP)-motif (Ser-Pro)_n (SP)_n as TC-His double-tag fusions were performed by transiently transforming tobacco and Arabidopsis leaves by Agrobacterium infiltration and also by stable transformation of Arabidopsis. These experiments revealed that SynGM expression and localization could be followed in live cells using the FlAsH-TC- or ReAsH-TC-tag in these plants. Also, more complex labeling protocols were achieved such as pulse-chase dual labeling with FlAsH and ReAsH for time-course experiments on a single SynGM type and co-localization experiments using green fluorescent protein (GFP)-fusion proteins (organelle markers) for referencing the expression of a SynGM labeled with ReAsH.

Materials and Methods

Reagents

The FlAsH and ReASH reagents were obtained from Invitrogen (Carlsbad, CA, USA) as Lumio™ Green In-Cell Detection kit and Lumio Red In-Cell Detection kit.

Plasmid Construction and Agrobacterium-Mediated Transformation

The SynGM constructs were cloned into a modified pET-28 vector (EMD Biosciences, La Jolla, CA, USA). All constructs for expression in plants were under transcriptional control of the 35S cauliflower mosaic virus promoter. The signal peptide sequence described by Shpak et al. (9) was placed 5′ to synthetic SacII-BamHI DNA fragments encoding (SP)₇ or (SP)₉ followed by a 6× His sequence and a TC domain (CCGPCC sequence). The gene construct was recombined into the plant destination vector pMDC32 using Gateway® technology (Invitrogen) (10). The plasmids were delivered into Agrobacterium tumefaciens (strain GV3101) by electroporation. Arabidopsis plants were stably transformed by the Clough and Bent method (11) using hygromycin as the selectable marker. Plants were dipped in Agrobacterium when most secondary bolts were 1–10 cm tall and carried multiple young floral buds (typically 5–8 days after clipping). Inoculations were performed by dipping aerial parts of the plants for a few seconds in 300 mL solution containing 5% (w/v) sucrose, 10 mm MgCl₂, resuspended Agrobacterium cells from a 150-mL overnight culture, and 0.03% (300 mL/L) of the surfactant Silwet L-77 (Lehle Seeds, Red Rock, TX, USA). After inoculation, plants were left in a low-light or dark location and covered with a transparent plastic dome to maintain humidity. The dome was removed, and the plants returned to the growth chamber 12–24 h after inoculation.

For transient expression, expanding leaves from 4-week-old Nicotiana benthamiana and Arabidopsis were inoculated with Agrobacterium delivered in the infiltration media [10 mM MgCl₂, 100 µM acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone), and 3% sucrose] (12). Inoculation was done using 1-mL syringes without needles by pressing the syringe tip against a lower leaf surface while slowly expelling enough liquid to form a 1-cm diameter wet spot in the leaf. Agrobacterium were grown on solid LB plates with kanamycin selection for 1–2 days, then cells were harvested and diluted into infiltration medium to an A_{600 nm} of 0.05–0.1 (tobacco) and 0.2 (Arabidopsis), and then incubated for 2 h before infiltration. Leaves were collected for imaging 2 and 6 days after infiltration.