A Scanning Electron Microscopy Technique for Viewing Plant-Microbe Interactions at Tissue and Cell-Type Resolution.

Observing pathogen colonization and localization within specific plant tissues is a critical component of plant pathology research. High resolution imaging, in which the researcher can clearly view the plant pathogen interacting with a specific plant cell, is needed to enhance our understanding of pathogen lifestyle and virulence mechanisms. However, it can be challenging to find the pathogen along the plant surface or in a specific cell type. Because of the time-consuming and expensive nature of high resolution microscopy, techniques that allow a researcher to find a region of pathogen colonization more quickly at low resolution and subsequently move to a high-resolution microscope for detailed observation are needed. Here we present paraffin scanning electron microscopy (PSEM), a technique in which paraffin embedded samples are first sectioned to identify a region of interest. Subsequently the same block is recut, deparaffinized, and used in scanning electron microscopy to generate high resolution images of plant-pathogen interactions in specific plant cell types. This method has several additional advantages over traditional SEM techniques, including reduced noise and better image quality. Here we use this technique to show that Fusarium oxysporum f. sp. lycopersici colonization is restricted in resistant Solanum pimpinellifolium, and that PSEM works well in additional pathosystems including maize leaves and Clavibacter michiganensis subsp. nebraskensis, and Arabidopsis leaves and Pseudomonas syringae.