Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4

Abstract

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido (Fic, Doc, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Fig. 1.. RIN4 coexpressed with AvrB exhibits a mass shift of +146 Da.

(A) Summary of protein mass shift of RIN4 or RAR1 coexpressed with AvrB in comparison with protein expressed alone. N/A not assessed. Proteins were expressed in BL21 (DE3). (B) Intact mass profile of RIN4 expressed alone or with AvrB (WT) or AvrB^Y65A. “*” symbols in black indicate mass peaks close to the theoretical mass, and “**” symbols in black indicate unknown modification peaks (+32 Da), which happened to be a signature mark for RIN4. “*” or “**” symbols in red indicate mass peaks with a shift of +146 Da. (C) Intact mass profile of RAR1 expressed alone or with AvrB or AvrB^Y65A. “*” symbols indicate mass peaks close to the theoretical mass, and “**” symbols indicate unknown modification peaks. (D) Intact mass profile of RIN4 coexpressed with AvrB^G46D, AvrB^R266A, or AvrB^D297A. “*” and “**” symbols indicate similar peaks as in (B).

Fig. 2.. Interaction between RIN4 and AvrB.

(A) Structure comparison of AvrB bound with ADP [Protein Data Bank (PDB) code: 2NUN] and with RIN4 C-NOI peptide (PDB code: 2NUD). Proteins, residues, and ADP are colored as indicated. Right: Electrostatic surface of AvrB with positive areas in blue, negative in red, and neutral in white (contour level: ±74 k_BT/e); RIN4 residues Y165, T166, and H167 are shown as sticks (side chain); ADP is shown as yellow sticks. (B) A. thaliana RIN4 structure model predicted by AlphaFold (AF-Q8GYN5-F1). Left: Structure model with the N-terminal and C-NOI domains indicated. Right: Model colored with confidence. (C) Amino acid sequence of RIN4 NOI domains with conserved motifs highlighted in red. (D) Pull-down assay for testing the interaction between AvrB and RIN4 (full-length, N-NOI, and C-NOI). (E) Pull-down assay for testing the interaction between AvrB and RIN4 (WT, T166A, T166E, and +146 Da). MW, molecular weight; BSA, bovine serum albumin.

Fig. 3.. AvrB modifies residue T166 of RIN4.

(A) LC-MS/MS analysis of RIN4 for identifying modification site of +146 Da. Chymotrypsin, trypsin, and Glu-C (or V8) proteases were used to digest RIN4 and generate various peptides for finding a minimal overlapping region that was modified. RIN4 residues built in the structure model (Fig. 2A) are colored in magenta. Peptides (with a total of 96 MS2 spectral counts or hits) around C-NOI have a minimum overlapping region (in cyan) containing residues G₁₆₄YTHIF₁₆₉. (B to D) Intact mass profile of mutant RIN4^T166A (B), RIN4^Y165A (C), or RIN4^H167A (D) expressed alone or coexpressed with AvrB. “*” and “**” symbols indicate similar peaks as in Fig. 1B.

Fig. 4.. The +146-Da mass increase of RIN4 is caused by rhamnosylation.

(A) Design of RIN4-Pcry construct. Residues are numbered as in native RIN4 protein. DrICE recognition motifs are colored blue with cutting position indicated by arrows. (B) Intact mass profiles of unmodified and modified Pcry peptides (purified after DrICE digestion of GST-RIN4^Pcry). “*” in black indicates the mass peak close to the theoretical mass. “*” in red indicates the mass peak with a shift of +146 Da. (C) Raw data of [M + 4H]⁴⁺ ions of Pcry peptides in intact mass analysis. Notable isotopic peaks of unmodified and modified peptides are shown. Mass shift (Δ mass in red) was calculated for each isotopic peak pair of unmodified and modified peptides. (D) Intact mass profile of RIN4 from in vitro reaction assay. RIN4 was incubated with AvrB and 100 μM cosubstrate (GDP-fucose or UDP-rhamnose). “*” and “**” symbols indicate similar RIN4 peaks as in Fig. 1B. (E) Model showing AvrB rhamnosylates RIN4 using UDP-rhamnose as cosubstrate. m/z,mass/charge ratio.

Fig. 5.. In vitro rhamnosylation of RIN4 catalyzed by AvrB.

(A) UDP-rhamnose (20 μM) hydrolysis catalyzed by AvrB (~6 nM) in the presence of GST-RIN4 (6 μM) as rhamnose acceptor. Reaction with AvrB (WT or mutant) was compared to buffer control. (B) In vitro rhamnosylation of RIN4 by AvrB with cosubstrate (100 μM) of ATP, glucose-6-P, GDP-fucose, or UDP-rhamnose. Rhamnosylated RIN4 was detected by immunoblotting (IB) using T166-Rha–specific antibody. “*” indicates degraded RIN4 in all immunoblotting images. (C) Effect of T166A or T166E mutation on rhamnosylation of RIN4 by AvrB (20 μM UDP-rhamnose as cosubstrate). (D) RIN4 rhamnosylation with UDP-rhamnose or dTDP-rhamnose as cosubstrate (2 μM). (E) Enzymatic activity test for AvrB mutants with UDP-rhamnose (2 μM) as cosubstrate. (F) Enzymatic activity test for AvrB mutants with dTDP-rhamnose (2 μM) as cosubstrate. (G) Coexpression of RIN4 and AvrB with BL21 (DE3) enzymes (RfbA, RfbB, RfbC, and RfbD) for producing dTDP-rhamnose in HEK 293 T/17 cells. Expression of AvrB and RIN4 was confirmed with anti–Strep-tag II antibody. (H) RIN4 rhamnosylation by AvrB in the presence or absence of Mg²⁺ (with 100 μM UDP-rhamnose). ns, not significant.

Fig. 6.. Catalysis mechanisms for RIN4 rhamnosylation by AvrB.

(A) Crystal structure of AvrB bound with RIN4 (8TXF). UDP-rhamnose atoms are numbered. (B) Crystal structure of AvrB bound with RIN4 and UDP-rhamnose (8TWS), representing the prereaction state. Possible hydrogen bonds (or polar contacts) are indicated by red dashed lines. Contacts between UDP and AvrB: O²′-G46, α-PO₄-R99, β-PO₄-R266, and β-PO₄-G267. Contacts between rhamnose and AvrB: O^0″-T166 (RIN4), O^0″-Y131, O^2″-A269, O^3″-D297, O^4″-T166 (RIN4), and O^4″-T125. (C) Crystal structure of AvrB bound with rhamnosylated RIN4 and UDP (8TWO), representing the postreaction state. (D) Crystal structure of AvrB^R266A bound with UDP (8TWJ). Contacts between UDP and AvrB: N³-N62, O²-H₂O, O²′-G46, O²′-H₂O, O³′-H₂O, α-PO₄-A269, α-PO₄-A270, α-PO₄-H₂O, β-PO₄-H₂O, β-PO₄-G267, and β-PO₄-R266. AvrB, RIN4, UDP-rhamnose, UDP, and rhamnose are colored as indicated in the figures with water molecules shown as red spheres.