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

SummaryThe 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.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (142 K)Download as PowerPoint slideHighlights► Bacterial papain-like protease AvrPphB cleaves kinases in host plant cell ► Activity-based probe for AvrPphB inspired on its unique substrate specificity ► Secretion of ProAvrPphB through narrow type III pilus requires prodomain ► Prodomain removal is essential to trigger cell death in resistant plants