Type III effector activation via nucleotide binding, phosphorylation, and host target interaction

Abstract

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

Figure 1. Structures of AvrB Complexed with RIN4_142–176 and ADP

(A) Difference electron density (F _o − F _c) from AvrB/RIN4_142–176 crystals centered on the C-terminal β-strand of RIN4_142–176 (green sticks) following molecular replacement with two AvrB molecules (PDB 1NH1). (B) Difference electron density (F _o − F _c) from AvrB crystals soaked with ADP following molecular replacement with 1 AvrB (PDB 1NH1). For (A) and (B), densities are contoured at 2 and 4 σ, respectively. (C and D) Ribbon diagrams of the AvrB/RIN4_142–176 and AvrB/ADP complexes, respectively. AvrB helices, strands, and loops are red, blue, and cyan, respectively, and the RIN4_142–176 ribbon is green. The overall structures are similar, and the different AvrB N and C termini between them likely reflects differences in their packing interfaces. (E and F) Surface representations of AvrB in the AvrB/RIN4_142–176 and AvrB/ADP complexes, respectively. (E) RIN4_142–176 is shown as a ribbon diagram with the side-chains of W154, Y165, T166, H167, and F169 represented as spheres to emphasize the surface grooves and concavities in AvrB that buries these residues. Note that the views represented in (C) and (E) and in (D) and (F) are identical.

Figure 2. Close-Up Views of the AvrB/RIN4_142–176 and AvrB/ADP Interfaces

(A and B) A semitransparent representation of the AvrB surface showing selected AvrB side-chains that contact RIN4_142–176 [green in (A)] and ADP in (B). Atoms of ADP and RIN4_142–176 side-chains are colored by type: C (green), N (blue), O (red), and P (pink). AvrB side-chains are coded by atom type; C (light blue), N (blue), and O (red). Positive and negative surface charges of the indicated AvrB side-chains are color coded blue and red, respectively. Text for AvrB residues that were mutated and tested for their ability to trigger RPM1 function are color coded as follows: red, full loss of function; yellow, partial loss of function; black, no loss of function. (C and D) A close-in semitransparent representation of the AvrB surface highlighting the functional requirement (complete, red; partial, yellow) for AvrB amino acids in triggering RPM1 function. Relevant side-chains and surfaces of AvrB residues are displayed as stick representations with color-coded labels as in (A) and (B). Stick representations of residues from RIN4_142–176 are labeled green in (C), and a stick representation of ADP is highlighted in (D).

Figure 3. ITC Data for Binding of RIN4 to Wild-Type AvrB and Selected AvrB Mutants

Fitted lines are shown for AvrB and mutants derivatives that did bind RIN4 (wild-type AvrB with full-length RIN4, wild-type AvrB with RIN4_142–176 and AvrB^QRY/AAA to RIN4_142–176). Raw data are displayed for AvrB mutants that did not bind RIN4_142–176. Lack of binding was verified by using 5× to 10× concentrations of RIN4_142–176 and AvrB variant relative to concentrations of wild-type components.

Figure 4. Interactions with RIN4 and ADP Are Required for AvrB Function in Activating RPM1

(A) Trypan blue staining of Arabidopsis Col-0 (RPM1) leaves 5 h after infection with Pto DC3000 expressing wild-type or mutant versions of HA-epitope tagged AvrB. EV is an empty vector (pBBR1 MCS-2) used as a negative control in Pto DC3000. QRY/AAA is a triple mutation of AvrB Q208, R209, and Y210 to alanine. Trypan blue staining of five infected leaves per construct was repeated twice in two independent experiments, and two representative leaves per AvrB mutant are presented. (B) Quantification of HR by electrolyte leakage (mean ± 2 SE) from leaves infected with Pto DC3000 expressing AvrB and mutant derivatives as labeled. The experiment was performed as in (A) and was repeated twice with similar results. Solid black lines represent the negative Pto DC3000(EV) (black triangles) and positive controls Pto DC3000(avrB-HA) (black diamonds). In the top graph, hatched lines represent mutations of AvrB residues making contacts with RIN4_142–176 from the crystal structure presented in Figure 2. In the bottom graph, dotted lines represent mutations of AvrB residues lining the ADP binding pocket, from the crystal structure presented in Figure 2. (C) Growth of Pto DC3000 expressing the wild-type or selected alanine mutant versions AvrB-HA on Arabidopsis Col-0(RPM1). White and black bars represent the bacterial populations at the time of inoculation (day 0) and 3 d postinoculation (day 3), respectively. Bars indicate the mean of four samples ± 2 SE and are representative of two independent experiments.

Figure 5. AvrB Is Specifically Phosphorylated in the Presence of Arabidopsis Extracts

(A) Amino acids critical for AvrB function are required for its phosphorylation in the presence of Arabidopsis extract. Autoradiograph of reaction mixtures containing Arabidopsis (Col-0) extracts and [γ-³²P]ATP with purified AvrB (36 kDa), indicated AvrB mutants, AvrB paralogs AvrC (39 kDa) and AvrXccC (36 kDa), or molecular weight markers (M). AvrB^R209A, AvrB^D297A, and AvrB^R266A include 14 additional amino acids from the recombination sequence of the Gateway cloning vectors at their C terminus (approximately 2 kDa). The lane labeled AvrB(−) does not contain plant extract. Coomassie brilliant blue staining of the radioactive gel, shown below, serves as a loading control. (B) Two surface representations of AvrB bound to RIN4_142–176 (left) and ADP (right). Red and yellow patches represent full and partial loss of AvrB function with respect to triggering RPM1, respectively. RIN4_142–176 is colored green, and amino acids making contacts with AvrB are highlighted (left). ADP is shown as a space-filled molecule (right). (C) AvrB residues contacting ADP are conserved among proteins of the AvrB protein family, but those contacting RIN4_142–176 are not. Amino acid sequence alignment of ten AvrB paralogs. Helices, strands, loops, and unstructured regions are denoted by red tubes, blue arrows, gray lines, and dashed gray lines, respectively. Secondary structure elements from residues 16 to 27 are rendered transparent, as they have been observed in the AvrB/ADP, but not in AvrB/RIN4_142–176 or free AvrB (PDB code 1NH1). Residues marked by green and yellow circles make contact with RIN4_142–176 or line the ADP-binding cavity, respectively. The paralogs used in this alignment are from P. syringae pv. glycinea (AvrB, AvrC), P. syringae pv. phaseolicola (AvrPphC, AvrB2, AvrB4–1, AvrB4–2), Xanthomonas campestris pv. campestris (AvrXccC), P. syringae pv. syringae (AvrB Psy1, AvrB Psy2, AvrB3).

Figure 6. Functionally Important Residues of AvrB Correspond to Catalytic Residues in Ser/Thr Protein Kinases

(A) A model of a possible ternary AvrB/RIN4_142–176/ADP complex based on the crystal structures of AvrB/ADP and AvrB/RIN4_142–176 (see Discussion). (B) The crystal structure of a ternary complex of cAMP-dependent kinase/inhibitor peptide/AMPPNP (PDB code 1CDK). The color scheme employed follows that of previous ribbon diagrams. RIN4_142–176 and inhibitory peptide are shaded green in (A) and (B), respectively. Atoms of ADP and RIN4_142–176 T166 in (A) and AMPPNP in (B) are colored by type: C (green), N (blue), O (red), and P (pink). Labeled residues are stick representations of those critical for AvrB/RIN4_142–176 or AvrB/ADP interaction in (A), and those critical for S17 phosphorylation, inhibited by S17A, as shown in (B).