Pharmacological approaches have made a tremendous impact on the field of microbial secretion systems. This protocol describes the inhibition of Golgi-dependent secretion in Magnaporthe oryzae though brefeldin A (BFA) treatment. State-of-the-art live-cell imaging allows tracking secreted proteins in their secretion pathways. Here we applied this protocol for defining the secretion systems of two fluorescently labeled effectors, Bas4 (apoplastic) and Pwl2 (cytoplasmic). Secretion of Bas4 is clearly inhibited by brefeldin A (BFA), indicating its Golgi-dependent secretion pathway. By contrast, secretion of Pwl2 is BFA insensitive and follows a nonconventional secretion pathway that is Snare and Exocyst dependent. The protocol is suitable to other plant-microbial systems and in vitro secreted microbial proteins.
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