Research Article

Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides

See allHide authors and affiliations

Science  12 Apr 2019:
Vol. 364, Issue 6436, eaav0748
DOI: 10.1126/science.aav0748

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

Glycosylation goes back and forth

Plants produce receptors that recognize fragments of microbial flagellin, thus monitoring for infection by bacteria. Buscaill et al. studied how a flagellin fragment is made accessible for recognition by host glycosidases, which degrade the glycosylations shielding the peptide that triggers the immune response. The pathogen, in turn, evades detection by altering flagellin glycosylation and inhibiting the host glycosidase. This aspect of plant defense against infection plays out in the apoplast, the extracellular space within plant tissues.

Science, this issue p. eaav0748

Structured Abstract


Immunogenic flagellin fragments are a signature of bacterial invasion in both plants and animals. Plants recognize flagellin fragments via flagellin sensitive 2 (FLS2), a model receptor kinase that is highly conserved among 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) contains hundreds of uncharacterized glycosidases and proteases.


We reasoned that pathogenic bacteria would suppress plant hydrolases that are important for immunity. To identify suppressed hydrolases in the apoplast of infected plants, we applied activity-based protein profiling with the use of chemical probes that irreversibly label the active site of hydrolases. We applied this strategy to study the infection of the tobacco relative Nicotiana benthamiana with the bacterial pathogens Pseudomonas syringae pv. tabaci (Pta6605), P. syringae pv. syringae (PsyB728a), and a virulent mutant of P. syringae pv. tomato [PtoDC3000(ΔhQ)].


Glycosidase activity profiling of apoplastic fluids isolated from PtoDC3000(ΔhQ)-infected plants revealed that the activity of β-galactosidase 1 (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 hrpR/S/L virulence regulators. Null mutants of N. benthamiana lacking BGAL1 generated by genome editing have substantially reduced apoplastic β-galactosidase activity and are more susceptible to PtoDC3000(ΔhQ), demonstrating that BGAL1 contributes to immunity. When investigating how BGAL1 functions in immunity, we discovered that treatment of PtoDC3000(ΔhQ) and Pta6605 bacteria with apoplastic fluids containing BGAL1 results in the release of an elicitor that triggers a burst of reactive oxygen species in leaf discs, a signature immune response in plants. The released elicitor is flagellin derived because the triggered immune response requires both the FLS2 receptor in the plant and the flagellin-encoding fliC gene in the bacteria. More precisely, treatment of purified flagella with apoplastic fluids containing BGAL1 facilitates the release of immunogenic peptides from flagellin.

The flagellin polymer of both PtoDC3000(ΔhQ) and Pta6605 is O-glycosylated with glycans consisting of several rhamnose residues and a terminal modified viosamine (mVio). Mutant Pta6605 bacteria carrying nonglycosylated flagellin, or carrying rhamnosylated flagellin lacking mVio, trigger the plant immune response when treated with apoplastic fluids, irrespective of BGAL1 presence, thus demonstrating that BGAL1 requires mVio for its function in immunity. Addition of a protease inhibitor cocktail to apoplastic fluids blocks the release of the flagellin elicitor from nonglycosylated flagellin, implicating apoplastic proteases in elicitor release acting downstream of BGAL1. Consistent with a specific role of BGAL1 in elicitor release, bgal1 null mutants of N. benthamiana show increased susceptibility only to bacterial strains carrying mVio. Treatment of PsyB728a with apoplastic fluids containing BGAL1 does not facilitate release of the flagellin elicitor because its flagellin carries a different glycan moiety lacking mVio, thus providing protection against recognition.


Glycosidase BGAL1 acts upstream of proteases in the apoplast of N. benthamiana to release immunogenic peptides from glycosylated flagellin, but only on glycosylated flagellin containing mVio. P. syringae strains use both BGAL1 inhibitors and glycan polymorphism to suppress BGAL1 function and escape recognition. Glycan polymorphism is common to bacterial pathogens, indicating a general role for flagellin glycans in evading recognition of bacterial pathogens by both plants and animals.

Control over hydrolytic release of immunogenic flagellin fragments.

Secreted β-galactosidase BGAL1 and proteases contribute to immunity against bacteria with glycosylated flagellin carrying terminal mVio (purple) in both Pta6605 (A) and PtoDC3000(ΔhQ) (B) by releasing immunogenic peptides (flg, red) that are perceived by the FLS2 immune receptor. PtoDC3000(ΔhQ) (B) mitigates BGAL1 activity by the production of a BGAL1 inhibitor, whereas PsyB728a (C) produces BGAL1-insensitive glycans (orange) to escape recognition.


Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.

View Full Text