Bacterial Glycosyrins Manipulate Plant Glycobiology Through A Novel Mechanism
The plant apoplast is a key battlefield during microbial infection. To avoid plant recognition, the model plant pathogen Pseudomonas syringae pv. tomato DC3000 secretes glycosyrins. These compounds inhibit the activity of the plant-secreted β-galactosidase BGAL1, thereby suppressing plant defense responses triggered by flagellin fragments.
On April 17, 2025, a research paper titled "Bacterial pathogen deploys the iminosugar glycosyrin to manipulate plant glycobiology" was published in Science by Renier A. L. van der Hoorn's team at the University of Oxford. This study reports for the first time the structure, biosynthesis, and multifunctional mechanism of action of glycosyrin. Using high-resolution cryo-electron microscopy and Chemical Synthesis, they revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde group, whose structure mimics that of a monosaccharide molecule. Glycosyrin biosynthesis is controlled by virulence regulators. Glycosyrins are ubiquitous in bacteria and can block flagellin recognition by hosts and alter the extracellular glycoproteome and metabolome of infected plants. These findings reveal a potential broad-spectrum mechanism by which plant pathogens manipulate their hosts through glycobiology.
To identify genes required for glycosyrin biosynthesis, the researchers transformed the lacZ gene encoding β-galactosidase from Escherichia coli into the PtoDC3000 ΔhopQ1-1 strain (the glycosyrin-producing wild-type strain in this study). This enzyme produces a blue color reaction with X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside). Subsequently, Tn5 transposon mutagenesis was performed, and glycosyrin-deficient mutants with an enhanced blue color were screened on virulence-inducing medium containing X-gal. The absence of glycosyrin was confirmed by a fluorescent substrate activity assay using purified LacZ, and transposon insertion sites were identified in 140 glycosyrin-deficient mutants. These Tn5 insertion sites were concentrated in four virulence gene regulatory factors (hrpR, hrpS, hrpL, and rhpS) and a putative glycosyrin biosynthesis gene cluster (gsn). A mutant lacking the gsn cluster (Δgsn) lost the ability to synthesize the Inhibitor, but transformation of this mutant with a plasmid carrying the gsn cluster restored inhibitor production. Transformation of the gsn cluster plasmid into E. coli also enabled glycosyrin synthesis. Therefore, the gsn gene cluster is essential for glycosyrin biosynthesis in bacteria.
To elucidate the molecular structure of glycosyrin, the researchers immobilized His-tagged LacZ on an affinity resin and used it to capture glycosyrin from crude secretion from a wild-type glycosyrin-producing strain until LacZ was saturated. Following imidazole washing and elution, a LacZ-glycosyrin complex with high inhibitor saturation was obtained. The structure of this complex was solved at 1.9 Å resolution using cryo-electron microscopy, and electron density was detected in the active site that was absent from the negative control Δgsn mutant secretome. This density revealed a five-membered ring structure with three well-defined chiral centers: two of which are putative hydroxyl groups and one is a putative branched geminal diol group, likely formed by hydration of an aldehyde group.
Glycosyrin binds to the enzyme active site, and the orientation of its hydroxyl groups closely mimics Galactose. The hydrated aldehyde groups enable the five-membered ring to mimic the conformation of a six-membered galactose ring. Furthermore, the protonated nitrogen of glycosyrin forms an electrostatic interaction with the conserved catalytic glutamate (E538) and a cation-π interaction with the aromatic tryptophan (W569). Similar interactions also occur in the active site of the β-galactosidase BGAL1. Inhibition experiments demonstrated that synthetic glycosyrin potently inhibited both LacZ and BGAL1, with IC50 values lower than those of two known six-membered ring iminosugar inhibitors (1-deoxy-galactonojirimycin and galactostatin).
To decipher the glycosyrin biosynthesis pathway, researchers first focused on the GsnB protein, as it has been shown to be specific for the gsn gene cluster in comparative genomic analyses. GsnB shares homology with RibD reductase, a riboflavin biosynthesis pathway. The GsnB structure predicted by AlphaFold2 contains a conserved active pocket similar to the RibD crystal structure, suggesting that GsnB may target similar substrates. This substrate likely also contains an amino group, as, unlike other gene clusters containing GsnA homologs, the gsn cluster lacks aminotransferase genes.
Given that glycosyrin is a pentose-like molecule, researchers hypothesized that 5-phosphoribosamine (PRA) could be a substrate for GsnB. Structural modeling also predicted that NADPH and PRA could bind to the GsnB active pocket. PRA is generated from 5-phosphoribosylpyrophosphate (PRPP) by PurF and is used for PurD-mediated Purine Biosynthesis. Experimental results confirmed that under purine supplementation, the ΔpurF mutant failed to produce glycosyrin, while the ΔpurD mutant was able to synthesize it. Notably, the ΔpurD mutant produced more glycosyrin than the wild type, likely because the loss of PurD makes more PRA available for glycosyrin biosynthesis. Therefore, the purine intermediate PRA is a precursor for glycosyrin biosynthesis. Based on the reductase activity of RibD, the researchers hypothesized that GsnB might similarly use NADPH to reduce PRA to 1-amino-1-deoxy-D-ribitol-5-phosphate (1ADRP). Subsequently, the putative phosphatase GsnC might remove the phosphate group from 1ADRP to produce 1-amino-1-deoxy-D-ribitol (1ADR). Finally, the putative oxidase GsnA might oxidize the secondary hydroxyl group of 1ADR to produce the ketose 5-amino-5-deoxy-L-ribulose. 5ADR can spontaneously convert to the detected glycosyrin.
To verify the biosynthetic steps, the researchers purified four Enzymes (PurF, GsnB, GsnC, and GsnA) and incubated them with PRPP and a cofactor. The reaction system produced a β-galactosidase inhibitor only in the presence of all four enzymes, demonstrating that these enzymes are sufficient for glycosyrin synthesis in vitro.
Through step-by-step reaction verification, the researchers sequentially generated each intermediate in vitro, then heat-inactivated the enzyme. The subsequent enzymes and cofactors were then added in the desired order, successfully synthesizing the inhibitor from the intermediates. GC-MS analysis revealed the detection of the corresponding intermediates (PRA, 1ADRP, and 1ADR) after each enzymatic reaction. Together, these results confirmed the glycosyrin biosynthetic pathway. The researchers then proposed a chemical transformation pathway for the terminal step of glycosyrin: the amino group of 5ADR reacts with the keto group to form a cyclic imine form, which then undergoes a spontaneous Heyns rearrangement to form the aldehyde structure, ultimately hydrating to form glycosyrin.
Fig. 1 The glycosyrin biosynthetic pathway branches off from the purine biosynthetic pathway. (Sanguankiattichai, et al. 2025)
The initially identified glycosyrin target was the β-galactosidase BGAL1, which participates in plant defense by releasing immunogenic peptides from glycosylated flagellin. Researchers discovered the presence of β-D-galactose-lectin proteins, known as RCAI, in the apoplast of the bgal1-1 mutant, indicating that BGAL1 is also involved in the processing of endogenous plant glycoproteins. Notably, wild-type plants inoculated with wild-type P. syringae showed significantly higher accumulation of RCAI-positive glycoproteins than those inoculated with the Δgsn mutant, whereas no such difference was observed in the bgal1-1 mutant. These results suggest that modifications of the host Glycoproteome are mediated by the inhibition of BGAL1 by glycosyrin.
In fact, during infection of Nicotiana benthamiana leaves and in the apoplast, glycosyrin also inhibits other β-galactosidases and β-glucosidases, as well as plant α/β-glucosidases. Therefore, glycosyrin is a multifunctional iminosugar produced by P. syringae, manipulating plant glycobiology by regulating diverse host glycosidases.
Fig. 2 Glycosyrin regulates multiple aspects of plant extracellular glycobiology. (Sanguankiattichai, et al. 2025)
Using high-resolution cryo-electron microscopy and chemical synthesis, researchers have confirmed that this glycosidase inhibitor, glycosyrin, is a unique iminosugar with a structure similar to galactose. The study found that virulence regulators induce glycosyrin biosynthesis during infection; that glycosyrin is produced via a unique iminosugar biosynthetic pathway; and that glycosyrin can also alter the Glycosylation of plant proteins and the accumulation of extracellular metabolites. Notably, glycosyrin is produced by a variety of plant-associated bacteria, suggesting that glycobiological regulation may represent a common battleground in plant-pathogen interactions.
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