Ultracut s ultramicrotome

Plants were grown as described for infection thread experiments. Large wild-type nodules (3–4 mm), the largest na-1 nodules (1–2 mm), and na-1 roots containing bacterial accumulations were fixed and stored in 2.5% glutaraldehyde in buffer (100 mM sodium phosphate at pH 7, 10 mM KCl, and 1 mM MgCl₂·6H₂O), washed twice in 0.1 M PIPES (Sigma Australia) buffer (pH 7), treated with 1% osmium tetroxide in 0.1 M PIPES buffer (pH 7) for 2 h, then washed twice and stored in 0.1 M PIPES buffer. Samples were then treated with 5% uranyl acetate (ProSciTech Pty Ltd, Australia) in 50% ethanol for 30 min, dehydrated through graded ethanol and propylene oxide, and embedded in Procure 812 resin (ProSciTech Pty Ltd) according to the manufacturer’s instructions. Sections 70 nm thick were cut with a Reichert Ultracut S Ultramicrotome, and images of cross-sections captured with an Olympus BX50 microscope (Supplementary Fig. S2). The sections were then collected onto copper grids, and stained with uranyl acetate and Reynolds Lead citrate, then imaged with a Hitachi HT7700 transmission electron microscope at 80 kV at ×1000–×15 000 magnification.

The stem apical meristems (SAMs) and the true leaves at 1 to 6 DAGs were collected and fixed in FAA (50% ethanol, 5% (v/v) acetic acid, 3.7% (v/v) formaldehyde) for at least 24 hours in the dark (Zhao et al., 2021 (link)). The fixed samples were then dehydrated gradually by treatment in sequential ethanol solutions with a gradient concentration of 30%, 50%, 70%, 80%, 90%, 95%, and 100%, and each treatment lasted 20 minutes. The dehydrated samples were immersed in acetone solution for 20 minutes twice and finally embedded in Eponate-12 resin (Ted Pella, Redding, CA, USA) (Cai et al., 2017 (link)). Sections of 3-micrometer thickness were cut using a Leica Ultracut Sultramicrotome and stained with 0.1% toluidine blue before imaging with a BX63 microscope (Olympus) under bright-field optics.