Methods for Assessing Plant Immune System Activation by Bacterial Extracellular Vesicles and Other Elicitors

Bacterial extracellular vesicles (BEVs) are released from the surface of bacterial cells and contain a diverse molecular cargo. Studies conducted primarily with bacterial pathogens of mammals have shown that BEVs are involved in multiple processes such as cell-cell communication, the delivery of RNA, DNA, and proteins to target cells, protection from stresses, manipulation of host immunity, and other functions. Until a decade ago, the roles of BEVs in plant-bacteria interactions were barely investigated. However, recent studies have shown that BEVs of plant pathogens possess similar functions as their mammalian pathogen counterparts, and more research is now devoted to study their roles and interactions with plants. In the following methods chapter, we provide five well-validated assays to examine the interaction of BEVs with the plant immune system. These assays rely on different markers or immune outputs, which indicate the activation of plant immunity (defense marker gene expression, reactive oxygen species burst, seedling inhibition). Furthermore, we offer assays that directly evaluate the priming of the immune system following BEV challenge and the effectiveness of its response to subsequent local or systemic infection. Altogether, these assays provide a thorough examination to the interactions of BEVs and the plant immune system.