Apreo s scanning electron microscope

Scanning electron microscopy was performed according to a modified method using an Apreo S scanning electron microscope (Thermo Fisher, Waltham, Massachusetts, United States) (Kang et al., 2006 ). For SG detection in the internode, paraffin sections were made as described by Li et al. (2009a (link),2009b (link)). The paraffin sections were stained with I₂‐KI and observed under a light microscope (Nikon DS‐U3; Nikon, Minato City, Tokyo, Japan) (Peng et al., 2014 (link)). For transmission electron microscopy analysis, leaves were treated in 3% glutaraldehyde and then fixed in 1% osmium tetroxide. After dehydrating in a gradient acetone series, the leaf sections were embedded in Epon812 medium for thin sectioning. Uranyl acetate and Reynolds' lead citrate were used to stain the thin sectioning, and an H‐600 IV transmission electron microscope was used to assess the picture (Hitachi, Chiyoda City, Tokyo, Japan) (Li et al., 2015 (link)).

Live bacteria in buffer C at a concentration of ~ 10⁸ bacteria/ml were applied as 5 μl drops to isopropanol cleaned silicon wafers. Following incubation for 20 min at room temperature (RT), bacteria were gently mixed with either 5 μl buffer C or 5 μl buffer C supplemented with 5 μM hGBP1_F, or hGBP1_F^R584‐586A, or hGBP1_F^R48A, and 2 mM GTP. Samples were fixed after 4 min and 45 min incubation time with 10 μl of 2 × concentrated fixative (8% formaldehyde, 4% glutaraldehyde in 1 × PBS) for 20 min. Samples were washed once with 1 × PBS and twice with purified water and then air dried. Dried samples were coated with gold for 200 s using a Desk V sputter coater (Denton). Scanning electron micrographs were acquired with an Apreo S scanning electron microscope (FEI, Thermo Fisher Scientific) operating at 2 kV.