Hydathodes are plant organs present on leaf margins of a wide range of vascular plants and are the sites of guttation. Both anatomy and physiology of hydathodes are poorly documented. We have recently reported on the anatomy of cauliflower and Arabidopsis thaliana hydathodes and on their infection by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) (Cerutti et al., 2017). Because hydathodes are natural infection routes for several pathogens, it is necessary to have a deep knowledge of their anatomy to further better interpret images of infected hydathodes. Here, we described different detailed protocols for gaining information on hydathode anatomy which are applicable to a wide range of plants (including monocots like barley and rice). Nomarsky and confocal microscopy were used to observe clarified thick samples. Optical microscopy in transmitted light and transmission electron microscopy were used to observed thin and ultrathin sections.

The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and stomatal biology. A critical limitation to methods used to observe plant-microorganism interactions in the pore is the application of too much pressure to the sample during observations and z-stack acquisitions. To solve this issue, we recommend the use of a long working-distance water immersion objective lens that allows observations even with thick samples.