A Substrate-Inspired Probe Monitors Translocation, Activation, and Subcellular Targeting of Bacterial Type III Effector Protease AvrPphB Original Research Article

The AvrPphB effector of Pseudomonas syringae is a papain-like protease that is injected into the host plant cell and cleaves specific kinases to disrupt immune signaling. Here, we used the unique substrate specificity of AvrPphB to generate a specific activity-based probe. This probe displays various AvrPphB isoforms in bacterial extracts, upon secretion and inside the host plant. We show that AvrPphB is secreted as a proprotease and that secretion requires the prodomain, but probably does not involve a pH-dependent unfolding mechanism. The prodomain removal is required for the ability of AvrPphB to trigger a hypersensitive cell death in resistant host plants, presumably since processing exposes a hidden acylation site required for subcellular targeting in the host cell. We detected two active isoforms of AvrPphB in planta, of which the major one localizes exclusively to membranes.