Crystal structure of the complex between Pseudomonas effector AvrPtoB and the tomato Pto kinase reveals both a shared and a unique interface compared with AvrPto-Pto

Abstract

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9A and of the AvrPtoB(121-205)-Pto complex at a resolution of 3.3 A. AvrPtoB(121-205) exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.

Figure 1.

Characterization of the Interaction between AvrPtoB121-2₀₅ and Pto. (A) A representative gel filtration experiment examining the complex between the Pto binding domain of AvrPtoB (residues 121 to 205) and full-length Pto. Aliquots of the gel filtration fractions were visualized by Coomassie blue staining following SDS-PAGE. The AvrPtoB_121-205 fragment was eluted in fractions 35 and 36 when incubated alone and in fractions 30 to 32 when incubated with Pto. MW, molecular weight standards (in kilodaltons) are shown on the left side. (B) Measurement of binding affinity between Pto and AvrPtoB_121-201 by ITC. Top panel: Raw ITC data. Eighteen injections of AvrPtoB_121-201 solution were added to the Pto protein solution in the ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for AvrPtoB_121-205–Pto interaction. The integrated heat is plotted against the molar ratio of AvrPtoB_121-205 added to Pto in the cell. Data fitting revealed a binding affinity of 1.1 μM.

Figure 2.

AvrPtoB_121-205 Has a Distinct Fold from That Present in AvrPto. (A) Structure of AvrPtoB_121-205, the domain required for interaction with Pto. The five α-helices in AvrPtoB_121-205 are labeled as A, B, C, D, and E. N, N terminus; C, C terminus. (B) Ile-181 in AvrPtoB forms extensive hydrophobic interactions with its neighboring residues for stabilizing the conformation of the rigid coil linking αC and αE. The side chains of certain AvrPtoB residues are shown in yellow. (C) The structure-based sequence alignment among AvrPtoB homologs (Lin and Martin, 2006). Identical amino acids are boxed in red; similar amino acids are boxed in yellow. The locations of the five α-helices are shown above the alignment. The program ClustalW was used for sequence alignment.

Figure 3.

Specificity Determinants for Recognition of AvrPtoB by Pto. (A) Overall structure of the complex between AvrPtoB_121-205 and Pto. AvrPtoB_121-205 and Pto are colored in pink and slate, respectively. Pto is shown as a surface representation. (B) Illustration of the overall structure of the complex between AvrPtoB_121-201 and Pto with the same orientation as in (A). The red frame highlights interface 1, and the blue frame highlights interface 2 of the complex. BC represents the loop linking helices B and C in AvrPtoB. (C) The detailed interactions around interface 1 highlighted in the red frame shown in (B). The side chains of AvrPtoB and Pto are shown in yellow (labeled in pink) and cyan (labeled in dark slate), respectively. Relevant amino acid residues are numbered, and a hydrogen bond is shown as a red dashed line. (D) The detailed interactions around interface 2 highlighted in the blue frame shown in (B). The side chains of AvrPtoB and Pto are shown in yellow and cyan, respectively. Relevant amino acid residues are numbered, and hydrogen bonds are shown as red dashed lines.

Figure 4.

Pto-Interacting Residues of AvrPtoB Are Important for Its Avirulence Activity. (A) Effects of substitutions in AvrPtoB_121-205 on the interaction with Pto as determined by gel filtration. Each AvrPtoB protein was mixed with full-length Pto and subjected to a gel filtration assay. Aliquots of the fraction corresponding to the peak of AvrPtoB_121-205–Pto complex were visualized by Coomassie staining following SDS-PAGE. (B) Effects of substitutions in AvrPtoB_1-307 on the interaction with Pto as determined by a Y2H assay. Blue patches indicate positive interactions. Protein gel blotting with anti-HA antibody in the bottom panel shows similar expression in yeast of wild-type AvrPtoB_1-307 and the mutant proteins. (C) Effects of point mutations in AvrPtoB_1-307 on its avirulence activity. Pst DC3000ΔavrPtoΔavrPtoB strains expressing wild-type AvrPtoB_1-307, AvrPtoB_1-307 mutants, or carrying an empty vector were vacuum inoculated into leaves of tomato RG-PtoR plants (Xiao et al., 2007). Bacterial populations in leaves were determined 0 and 4 d after inoculation. Experiments were repeated twice with similar results. Protein gel blotting with anti-AvrPtoB antibody in the bottom panel shows that all of the proteins were secreted at similar levels from Pst. cfu/cm² = colony-forming units per centimeter².

Figure 5.

Pto Substitutions L205A/F213A Disrupt Interaction of Pto with AvrPtoB but Not AvrPto. (A) Effects of substitutions in Pto on its interaction with AvrPtoB_1-307 and AvrPto as determined by a Y2H assay. Blue patches indicate positive interactions. Protein gel blotting with anti-HA antibody in the bottom panel shows equal expression of wild-type Pto and derived mutants in yeast. (B) Effects of Pto substitutions H49E/V51D on Pto interaction with AvrPtoB_1-307 or AvrPto as determined by a Y2H assay. (C) Effect of Pto mutations on effector-elicited cell death in N. benthamiana leaves. Agrobacterium-mediated transient coexpression of wild-type Pto or derived mutants together with AvrPtoB_1-307 or AvrPto in N. benthamiana leaves. AvrPto alone causes weak cell death in this assay (see vector control), but much stronger cell death is apparent when AvrPto is coexpressed with wild-type Pto or the Pto K215D and V242D proteins. Note that the wild-type Pto, Pto K215D, or Pto V242D proteins, when expressed alone (without an effector protein), do not cause cell death (see Figure 6A).

Figure 6.

The Pto^L205A/F213A Protein Has Prf-Dependent CGF Activity. (A) Agrobacterium-mediated transient expression of Pto^L205A/F213A in N. benthamiana leaves caused rapid cell death independent of AvrPto or AvrPtoB. Leaves were inoculated with Agrobacterium strains that deliver T-DNAs encoding the Pto variant proteins with the amino acid substitutions indicated. Expression of the Pto variant proteins in N. benthamiana leaves was confirmed by protein blots as shown at the bottom using an anti-HA antibody. (B) Prf is required for hypersensitive response triggered by Pto^L205A/F213A. N. benthamiana plants were subjected to virus-induced gene silencing using a tobacco rattle virus (TRV2) construct carrying a fragment of Prf. The top panel shows a control leaf from a plant infected with TRV alone. The bottom panel shows a leaf silenced for the Prf gene.

Figure 7.

The Active Conformation of Pto Is Important for Pto Interaction with AvrPtoB_121-205. (A) Pto P+1 loop adopts a similar conformation in its complex with AvrPto or AvrPtoB_121-205. Superimposition of Pto around the P+1 loop region from AvrPtoB_121-205-Pto (slate) and AvrPto-Pto (pink) complexes. (B) Pto T199 in AvrPtoB_121-205-Pto complex is phosphorylated as indicated by electron density. Omit electron density map around Pto^T199 (shown at 1.2 sigma). The map was calculated using the CNS program. Hydrogen bonds are represented by red dashed lines. Pto^pT199 was not used for calculation of the electron density map. (C) Effects of various Pto substitutions on the interaction of Pto with AvrPtoB_121-205. Gel filtration was used to assay the interaction of Pto proteins and AvrPtoB_121-205 as described in Figure 1A.

Figure 8.

Structural Comparison of the AvrPtoB_121-205-Pto and AvrPto-Pto Complexes. Superimposition of the AvrPtoB_121-201-Pto and AvrPto-Pto complexes. The red and blue frames highlight the two unique interfaces in the complexes, with red indicating the AvrPtoB_121-205-Pto interface 1 involving contact of the helix D in AvrPtoB with loop L1 and helix α1 in Pto and blue indicating the AvrPto-Pto unique interface involving H49/V51 from the loop preceding β1 in Pto. The pink frame indicates the shared interface between AvrPtoB-Pto and AvrPto-Pto complexes. “BC” with a sequence AVAF and “CD” with a sequence GINP represent the two loops in AvrPtoB and AvrPto, respectively, that are involved in the interaction with the P+1 loop of Pto. Pto (shown in slate in AvrPtoB_121-205-Pto and deep salmon in AvrPto-Pto) from both complexes was used for structural superimposition.