The green fluorescent protein (GFP)
coding region was cloned as a glutathione S-transferase (GST) tagged recombinant gene. GFP was amplified from pDH51-GW-EGFP (GenBank: AM773753.1) using the following forward and reverse primers: 5′- GGC TCG AGA TGG TGA GCA AGG GCG AGG AG-3′
and 5′- GGA AGC TTT CAC TTG TAC AGC TCG TCC ATG CC
-3′. The PCR product was digested with XhoI and HindIII and cloned into pGEX-KG 
Preparation of E. coli Expressing GFP
E. coli strain Bl21 (DE3) (Novagen) carrying the plasmid pGTf2 (TAKARA BIO INC) was transformed with pGSTGFP. Recombinant E. coli cells were selected on LB plates containing 200 µg ml−1 ampicillin and 35 µg ml−1 chloramphenicol. A single colony was used to inoculate a pre-culture containing 20 ml of LB supplemented with ampicillin (200 µg ml−1) and chloramphenicol (35 µg ml−1). The pre-culture was grown overnight at 37°C and used to inoculate 1 l of LB supplemented with ampicilin and chloramphenicol. E. coli was grown at 37°C to a cell density of 0.6 to 1 A600 units. Cells were cooled down on ice and 1 mM of Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induce expression of GFP. After incubation on a shaker (160 rpm) for 16–20 hours at 18°C, cells were harvested by centrifugation and washed twice with 1 l of 5 mM MES pH 5.8 (wash buffer) and resuspended in wash buffer. Cultures were used immediately.
Preparation of Saccharomyces cerevisiae Expressing GFP
GFPyeast clone TDH3 (YGR192C) (Invitrogen, California, USA) expressing glyceraldehyde-3-phosphate dehydrogenase fused to GFP was selected among the entire GFPyeast clone library for its high emission of green fluorescence (yeastgfp.yeastgenome.org). A single colony was used to inoculate 1 l of Yeast-extract Peptone Dextrose (YPD) liquid media and the culture was grown for 48 h at 28°C. Cells were harvested by centrifugation, washed twice with 1 l of wash buffer and re-suspended in wash buffer. Cultures were used immediately.
Plant Growth Conditions
Arabidopsis (Arabidopsis thaliana
[Col-0]) plate culture: Seeds were germinated axenically on Petri dishes containing Murashige and Skoog (MS
) medium solidified by 3.2 g l−1
of phytagel (Sigma). Plates were positioned vertically so that germinating radicals grow downward along the gel surface. Plants were grown for 2–3 weeks in a growth room with 16/8 h light/dark, 21°C, 150 µmol m−2
light intensity Arabidopsis axenic hydroponic culture: sterile seeds were sown in agar-filled 1.5 ml microcentrifuge tubes without cap and bottom. Microcentrifuge tubes were filled with 1.5 ml agar (0.68%) and tube bottoms cut off after agar had solidified, standing in a rack holder, and placed into sterile Combiness boxes (Microbox, Belgium) contained 300 ml half-strength MS medium. Adding 1 Arabidopsis seed into each tube, the boxes were incubated in a cold room for three days and then transferred to a growth cabinet (21°C, 16 h/8 h day/night, 150 µmol m−2
). Plant roots grew from tubes into the solution. The boxes were aerated from day 11 after sowing by pumping air through a sterile filter (0.22 µm Millipore Filter, Ireland). Plants were grown for another 20 days and then in N-free MS medium for 3 days. Then 20 ml of GFPE. coli
30) was added for 24 h. Plant incubated with 20 ml of wash buffer were used as a control. Plant were harvested, rinsed in deionized water, and immediately submersed in liquid N2
and stored at −80°C.
Tomato (Solanum lycopersicum) vermiculite culture: seeds were geminated in soil for 10 days prior to being transferred into 200 ml pots containing vermiculite (one seedling per pot) in a growth room (16/8 h light/dark, 21°C, 150 µmol m−2 s−1). Pots were watered daily with tap water with addition of fertilizer (N-P-K: 15-15-15) once a week. Plants were grown for 2 to 3 weeks until shoot size was 10–15 cm.
Tomato hydroponic culture: 8–12 cm tall plants grown on vermiculite were carefully transferred into hydroponic culture consisting of 0.5 l water at pH 5.8 supplemented with 10 µM CaSO4 (hydroponic solution) with 1 seedling per pot. The hydroponic cultures were continuously aerated and mixed by gentle stirring with a magnetic stirrer bar.
Uptake of E. coli and Yeast by Roots of Arabidopsis and Tomato
To assess uptake of E. coli and yeast by Arabidopsis, 5 ml of GFPE. coli or GFPyeast preparation (see above) at a cell density of 2 A600 units was carefully added to roots of plants grown axenically on MS plates (see above) and incubated for 4 h horizontally at room temperature. Plants were carefully removed from the medium and roots washed with deionized water before being analyzed by confocal laser microscopy (CLSM, see details below). To assess uptake of E. coli and yeast by tomato in hydroponic cultures, plants were initially grown in hydroponic solution for 3 days to ensure the integrity of the roots. 20 ml of GFPE. coli or GFPyeast preparation at a cell density of 50 A600 units was then added into the 500 ml hydroponic culture. After an overnight incubation at room temperature, roots were washed with deionized water and analyzed by CLSM.
For analysis of Arabidopsis and tomato root section by CLSM, visually assessed roots regions showing high fluorescence were excised (5–10 mm long), washed and embedded in 3% agarose. Hand-cut cross sections were transferred into curved slides, washed thoroughly with deionized water and analyzed by CLSM (see below). For analysis of tomato root sections by TEM, root regions showing high uptake by CLSM were coated with agarose before processing to ensure that bacteria external to roots were trapped in the agarose and not dislodged during cutting.
Time Course Experiment to Assess Status of GFPyeast in Tomato Roots
Ten tomato plants grown hydroponically for three days in hydroponic solution (see above) were incubated with GFPyeast overnight. Roots were carefully rinsed with deionized water and plants were placed in fresh hydroponic solution. The hydroponic solution was replaced every two days. Duplicate plants were removed from hydroponic culture at different time points and roots were treated with hydrogen peroxide (15%, 10 min) to sterilize the root surface. Roots from one plant were analyzed by CLSM and roots from the other were ground in liquid N2 and analyzed for TDH3:GFP content by western blotting.
Entire tomato roots were ground in liquid nitrogen and resuspended in 0.5 ml of 50 mM Tris (pH 7.5) supplemented with 0.1% Tween20. Non-soluble material was discarded by centrifugation at 14,000 rpm for 30 min. Total protein content of the extracts was determined as described by Bradford 
. Equal amounts of protein sample was resolved by SDS-PAGE and characterized by western blot analysis using anti-GFP antibody (0.4 µL ml−1
, Roche) as primary antibody and Alexa Fluor 680 goat anti-mouse (Molecular Probes) as secondary antibody. Detection was performed with an Odyssey infrared imaging system (Li-COR, USA).
15N-Labeling of E. coli
N-labeling of E. coli
Bl21 cells was carried out as described by 
N-labeled E. coli
cells (0.5 l) were harvested by centrifugation and washed four times with 0.5 l of deionized water. E. coli
cells were then re-suspended in 1 l of water and used immediately for the incubation experiment.
Uptake of 15N-Labeled E. coli by Tomato
Twenty-one tomato plants (15 days old) were grown for three days in hydroponic solution (see above). Seven plants were incubated in 1 l of 15N-labeled E. coli solution for 1 h. After incubation, roots were gently rinsed with deionized water and plants were transferred to hydroponic solution. During this process, special care was given to avoid any contamination of the shoots by the bacterial solution. The remaining 15N-labeled E. coli incubation solution was centrifuged (3000 rpm, 15 min) and the supernatant sterilized by filtration (0.22 µm Millipore Filter, Ireland) to remove remaining E. coli cells. Seven plants were incubated for 2 h in the filtered supernatant (“control 2”). After incubation, roots were gently rinsed with sterile deionized water and plants were further grown in hydroponic solution. A further seven plants were grown in hydroponic culture without addition of E. coli (“control 1”). All plants were grown for a further 2 weeks with hydroponic solution changed daily. Subsequently, 2–3 new leaves of each plant were excised and dried at 60°C overnight, weighted and homogenized. The samples were analyzed for total nitrogen (N) and 15N content with continuous flow Isotope Ratio Mass Spectrometer (IRMS, Stable Isotope Facility, University of California, Davis).
Total RNA of Arabidopsis roots grown in hydroponic culture were extracted using NucleoSpin® Plant Kits (BD Biosciences Clontech, Japan). RNA of plants incubated with or without E. coli (control) were labelled with Cy3 or Cy5 fluorescent dye, mixed and used for subsequently hybridization onto 4x44K Agilent Arabidopsis GeneChip arrays (Agilent Technologies, USA). Labelling and hybridization of RNA, including scanning of the chips were performed by the Australian Genome Research Facility (AGRF, Victoria, Austrlia). Expression values (log10) for three biological replicates were extracted using robust multi-array analysis with perfect match correction and quantile normalization. Genes with ≥3 fold change were computed using one-way ANOVA (p<0.05) with Partek Genomics suite.
The microarray hybridization data have been submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo
) under accession number GSE22277.
Roots of Arabidopsis and tomato incubated with GFPE. coli
yeast were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 6.8 overnight at 4°C. After washing in 0.1 M phosphate buffer, roots were dehydrated through a graded ethanol series and infiltrated with LR White Resin and polymerized overnight at 50°C. Thin sections were cut with a Leica Ultracut UC6 ultramicrotome, picked up on carbon coated copper grids, stained with uranyl acetate and Reynold's lead citrate 
and viewed in a JEOL 1010 transmission electron microscope operated at 80 kV and images were captured on a Olympus Soft Imaging Solutions Megaview III digital camera.
Thin sections were labeled using an anti-GFP antibody (Clontech, Mountain View, USA) as the primary antibody and a goat anti-mouse secondary labeled with 10 nm colloidal gold (British Biocell International, Cardiff, UK). Sections were also labeled with cellulase gold, made according to 
. The cellulase was 1,4-(1,3:1,4)-β-D-Glucan 4-glucano-hydrolase from Trichoderma reesei
(Sigma Aldrich, St Loius, USA). As a control, root sections were exposed to 2 mg ml−1
cellulase for 16 h prior to labeling.
Cellulase Activity Analysis
26-days old hydroponically grown Arabidopsis were incubated with E. coli Bl21 overnight. Plants not incubated with E. coli were used as negative control. Roots were rinsed twice in fresh medium and then transferred to fresh medium containing 31 µg ml−1 resorufin-β-D-cellobioside (Res-CB) (Marker Gene Technologies Inc., Eugene, OR, USA), a long-wavelength fluorescent substrate, which releases red fluorescent fluorophore resorufin upon cleavage. Roots were incubated for 2 h at room temperature, washed and inspected under CLSM.
A Zeiss LSM510 META (Carl Zeiss, Germany) confocal laser scanning microscope (CLSM) was used with 10x dry, 20x water immersion objectives, 40x and 60x oil immersion objectives. GFP and Res-CB were visualized by excitation with an argon laser at 488 nm and HeNe1 laser at 543 nm; detection with a 505–530 nm and 560–615 nm band-path filter, respectively.