Stomatal Bioassay to Characterize Bacterial-Stimulated PTI at the Pre-Invasion Phase of Infection.

Bacterium-triggered stomatal closure is a functional output of plant immunity, also known as stomatal defense. This is an early response mediated by the recognition of pathogen-associated molecular patterns (PAMPs) by the plant's pathogen recognition receptors (PRRs). Several approaches to analyzing stomatal movement in response to bacteria have been described, but difficulties in fine-tuning the experimental procedures still exist. Here we provide a detailed method for assessing stomatal defense via high-quality microscopic imaging and explain trouble-shooting steps to obtaining robust data. Although this procedure requires minimal manipulation of the leaf sample, it is crucial to control all environmental conditions and extrinsic variables that could interfere with stomatal movement. The method described here is also suitable for in vivo characterization of stomatal response in new pathosystems and can be used in conjunction with other profiling assays to gain a detailed understanding of early PAMP-triggered immunity (PTI).