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Ichinose and Renier A. L. van der Hoorn INTRODUCTION: Immunogenic flagellin fragments are a signature of bacterial invasion in both plants and animals. Plants recognize flagellin fragments via FLS2, a model receptor kinase that is highly conserved amongst angiosperms. However, little is known about events upstream of flagellin perception by FLS2. The flagellin fragments recognized by FLS2 are buried in the flagellin polymer structure and require hydrolytic release before recognition can occur, yet the hydrolases releasing these elicitors remain to be identified. Uncovering their identity is a daunting task because the extracellular space of plants (the apoplast) is known to contain hundreds of uncharacterized glycosidases and proteases. RATIONALE:We reasoned that pathogenic bacteria would suppress plant hydrolases that are important for immunity. We therefore monitored the activity of apoplastic hydrolases using activitybased protein profiling (ABPP) using chemical probes that irreversibly label the active site of hydrolases in an activity-dependent manner. We applied this strategy to study the infection of the tobacco relative Nicotiana benthamiana with the model bacterial pathogens Pseudomonas syringae pathovars tomato DC3000 (PtoDC3000); tabaci (Pta6605); and syringae (PsyB728a). RESULTS:Glycosidase activity profiling of apoplastic fluids isolated from PtoDC3000-infected plants revealed that the activity of the β-galactosidase BGAL1 is suppressed in the apoplast during infection.BGAL1 suppression is caused by a heat-stable, basic, small inhibitor molecule that is produced by the bacteria under the control of the hrpR/S/L virulence regulators. Null mutants of N. benthamiana lacking BGAL1 generated by genome editing are more susceptible, demonstrating that BGAL1 contributes to immunity. When studying why BGAL1 is suppressed, we discovered that treatment of PtoDC3000 and Pta6605 with apoplastic fluids containing BGAL1 releases an elicitor that triggers the release of reactive oxygen species (ROS) in leaf discs, a signature immune response in plants. The released elicitor is flagellin-derived because the ROS burst requires both the FLS2 receptor in the plant and flagellinencoding fliC in the bacteria. The flagellin polymer of both PtoDC3000 and Pta6605 is O-glycosylated with a glycan consisting of several rhamnose residues and a terminal modified viosamine (mVio).Treatment of mutant Pta6605 bacteria carrying nonglycosylated flagellin triggers the ROS burst when treated with apoplastic fluids, even in the absence of BGAL1, demonstrating that BGAL1 requires

One-sentence summary This review discusses and illustrates nine extracellular strategies that plant pathogens and symbionts use to avoid the recognition by cell surface receptors. Recognition of microbe-associated molecular patterns (MAMPs) by cell-surface receptors is pivotal in host-microbe interactions. Both pathogens and symbionts establish plant-microbe interactions using fascinating intricate extracellular strategies to avoid recognition. Here we distinguish nine different extracellular strategies to avoid recognition by the host, acting at three different levels. To avoid the accumulation of MAMP precursors (level-1), microbes take advantage of polymorphisms in both MAMP proteins and glycans, or downregulate MAMP production. To reduce hydrolytic MAMP release (level-2), microbes shield MAMP precursors with proteins or glycans and inhibit or degrade host-derived hydrolases. And to prevent MAMP perception directly (level-3), microbes degrade or sequester MAMPs before they are perceived. We discuss examples of these nine strategies and envisage three additional extracellular strategies to avoid MAMP perception in plants.