Wga alexa fluor 488

Jurkat T cells (ATCC TIB-152) were grown in RPMI (Sigma-Aldrich, Madison, WI). The culture media was supplemented with 10% fetal calf serum (FCS) (PAA), 10 mM HEPES (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM L-glutamine, and antibiotics [50 units penicillin, 50 μg streptomycin and 100 μg neomycin per mL] (Sigma-Aldrich). Cells were incubated at 37 °C with 5% CO₂. Labeling was done by resuspending 1 mL of cells in a solution of Nuclear Mask DeepRed [1 in 200 dilutions from stock (250× concentrate in DMSO)] (H10294, ThermoFisher) and WGA-AlexaFluor 488 [10 μg/mL concentration] (W11261, ThermoFisher). They were then incubated for 15 minutes at 37 °C before being washed 3x in PBS. For fixation, cells were resuspended in 4% PFA and incubated at 37 °C for 15 minutes, before being washed 3x in PBS. 3D stack was recorded in each color channel using a Zeiss LSM 900 confocal in confocal scan mode (voxel size: 0.62 × 0.62 × 0.54 μm (xyz)).

In vitro tests were performed with representatives of each clade obtained by the analyses of internal transcribed spacer (ITS) sequences of the fungal isolates (Figures 1, 2) using leek (Allium porrum), a generally used host plant in DSE resynthesis experiments (see Mandyam and Jumpponen, 2008 (link); Knapp et al., 2012 (link)) to test the basic symbiotic nature of the fungi. Five replicates for each fungal isolate and five control plants were incubated in each series according to Knapp et al. (2012) (link). The fungus and isolates of its clade were considered a root endophyte if it colonized the roots without symptoms.
Root samples from the field and in vitro experiments were studied microscopically. The cleared roots were stained using the fluorescence labeled lectin, WGA-AlexaFluor488 (Wheat Germ Agglutinin, Alexa Fluor 488 conjugate, Molecular Probes W11261, Thermo Fisher Scientific, Lithuania), a cell-wall-specific dye used for in planta visualization of fungal endophytes (e.g., Andrade-Linares et al., 2011 (link)). Root samples were examined using a light microscope with Nomarski (differential interference contrast, DIC) optics and a Nikon Eclipse 80i microscope equipped with a Spot 7.4 Slider camera (Diagnostic Instruments, Inc.), differential interference contrast (DIC), and a filter wheel with excitation and emission filters for visualization of Alexa Fluor 488 probe.

IT counts were performed using a Zeiss Axioplan 2 fluorescence microscope using the Zeiss Plan-Neofluar 20x/0.5 objective lens with excitation at 561 nm and an emission filter at 580 to 660 nm. Whole roots were mounted on microscope slides and IT counts performed on a per-root basis. Hairy roots expressing promoter-GUS constructs were GUS-stained as previously described [21 (link)]. GUS expression at the whole root level was observed using a Zeiss Discovery V8 stereo microscope. For concomitant visualisation of GUS-promoter activity and AM colonisation, GUS-stained roots were cleared in 10% KOH for 10 min and counterstained with 1 μg/ml WGA-Alexa Fluor 488 (Thermo Fisher, W11261). Bright field and fluorescent images were captured using a Zeiss Axioplan 2 fluorescence microscope with overlaying of the images performed using Fiji ImageJ [73 (link)]. For quantification of AM colonisation, roots were stained with 5% black ink (Noir de Jais, Shaeffer) according to [74 (link)]. Whole roots were mounted on microscope slides, and the frequency of arbuscules, fungal hyphae, and vesicles were counted as previously described [75 (link)].

Fungal structures in Medicago roots were stained with WGA-Alexa fluor 488 (Thermo Fisher Scientific) as described previously [51 (link)]. The root fragments were selected randomly and examined microscopically using an Olympus SZX16 stereomicroscope (Olympus, Tokyo, Japan). The colonization efficiency was evaluated by the grid method [64 (link)].

T. virens root colonization of maize seeds was examined using confocal microscopy (Fluoview FV10i, Olympus, Tokyo, Japan). For this analysis, transverse free-hand sections of maize roots were prepared. After 5 d post inoculation (d.p.i) maize roots were collected and either washed in phosphate-buffered saline (PBS) pH 7.4 or fixed in fresh ethanol: acetic acid (3:1, v/v) solution. Two staining methods were used: for fresh tissues a wheat germ agglutinin (WGA)-Alexa Fluor™ 488 (Thermo Fisher Scientific, MA, USA) /FM4-64 dye (Thermo Fisher Scientific, USA) mixture was used, while fixed tissues were stained with WGA-Alexa Fluor™ 488/Propidium iodide (PI) (Sigma-Aldrich, USA) mixture. Fungal material was stained with WGA-Alexa Fluor™ 488 (Mochizuki et al., 2011 (link)). Plant cell walls were stained with PI, while the plasma membranes were stained with FM4-64 (Bolte et al., 2004 (link)).
For fixed tissues, roots were treated with 10% KOH for 4 h at 95°C and then transferred to PBS pH 7.4 for 1 h. Samples were infiltrated with the staining solution (20 μg/mL PI; 10 μg/mL WGA-Alexa Fluor™ 488, 0.02% Tween 20 made up in 1X PBS) for 15 min twice. Samples were distained in PBS-tween (0.02%) and stored in the dark at 4°C. For fresh tissues, samples were washed in PBS solution and infiltrated with the same staining solution mentioned above, except that PI was substituted for 5 mM FM4-64.

The cell walls of the root cells were stained with propidium iodide, as previously described^{35 (link)}. To stain the components of the secondary cell wall, the Arabidopsis seedlings were washed in sterilized water and incubated in 1 μg/mL WGA-Alexa Fluor 488 (ThermoFisher Scientific, Waltham, MA, U.S.A.) in sterilized water at 25 °C for 1 h and washed five times for 30 min for each wash. Lignin and suberin were stained with basic fuchsin and Auramine O, respectively, as described by Ursache et al.^{31 (link)}.