Background

Environmental cues are important factors in determining the outcome of host-pathogen interactions. The disease triangle paradigm requires, among all, a favorable environment for disease to develop (Francl, 2001; Scholthof, 2007). High humidity, for example, represses HR development and negatively regulates ETI (Zhou et al., 2004; Xin et al., 2016; Mwimba et al., 2018). It is, thus, important to quantify the effect of environments on host-pathogen interactions.

Cell death by HR during ETI is often quantified using a time-course measurement of ion leakage from infected tissue to the aqueous solution surrounding the tissues (Hatsugai and Katagiri, 2018). However, when interested in assessing the contribution of the environment to the HR development, it is necessary for tissue to remain in the environment being studied until HR has developed.

In this protocol, we subject infected tissue to 50% RH or 90% RH for 36 h before conductivity is measured. Also, we have adjusted the dosage of the pathogen to OD_600nm = 0.002 to delay the onset of HR and maximize the effect of the environment on HR development. The method adapted here was originally designed to measure cell death in senescing leaves (Woo et al., 2001). Leaves were immersed in 400 mM mannitol, and conductivity was expressed as percentage of the ratio of conductivity measurement before and after boiling. In this modified method, we use tissue of equal size (leaf disc), which make reporting percent ion leakage optional. This protocol was used in our study (Mwimba et al., 2018) and can be applied to other environmental cues or to chemical treatments.