Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides

Abstract

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

Overexpression of F-Box Nictaba Promotes Defense and Anthocyanin Accumulation in Arabidopsis thaliana After Pseudomonas syringae Infection

“…Plants and animals need to recognize bacterial flagellin to trigger defense responses. For example, Nicotiana benthamiana cells secrete the enzyme β-galactosidase 1, which targets terminally modified viosamine that is part of the flagellin O-glycan on Pseudomonas syringae pathovars (Buscaill et al, 2019). As such, these glycan structures on flagella are important for host immunity to bacterial pathogens.…”

Dissection of the Complex Transcription and Metabolism Regulation Networks Associated with Maize Resistance to Ustilago maydis

“…On one hand, plant can recognize the pathogen-associated molecular patterns (PAMP) of pathogens to subsequently activate PAMP-triggered immunity (PTI) [2]. On the other hand, pathogens could evade PTI through PAMP modification and secreted effectors [3][4][5]. Furthermore, effectors could be perceived by host-resistance (R) proteins, such as nucleotide-binding leucine-rich repeats (NB-LRR) proteins, which subsequently activate more robust and rapid defense response, so-called effector-triggered immunity (ETI), often leading to hypersensitive response (HR) or programmed cell death (PCD) in the host [6].…”

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae

“…In plants, bacterial flagellin fragments are sensed by the FLS2 receptor, unless they are properly shielded by glycosylation. To “visualize” the flagellin glycopeptides, the host produces glycosidases such as BGAL1 to strip the protective sugar residue at the nonreducing ends ( 49 ). As a defense strategy, the MOP flippases in pathogens likely serve as a molecular checkpoint to ensure that the vulnerable glycans remain inside the cytoplasm.…”