The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation

Abstract

The major virulence strategy of phytopathogenic bacteria is to secrete effector proteins into the host cell to target the immune machinery. AvrPto and AvrPtoB are two such effectors from Pseudomonas syringae, which disable an overlapping range of kinases in Arabidopsis and Tomato. Both effectors target surface-localized receptor-kinases to avoid bacterial recognition. In turn, tomato has evolved an intracellular effector-recognition complex composed of the NB-LRR protein Prf and the Pto kinase. Structural analyses have shown that the most important interaction surface for AvrPto and AvrPtoB is the Pto P+1 loop. AvrPto is an inhibitor of Pto kinase activity, but paradoxically, this kinase activity is a prerequisite for defense activation by AvrPto. Here using biochemical approaches we show that disruption of Pto P+1 loop stimulates phosphorylation in trans, which is possible because the Pto/Prf complex is oligomeric. Both P+1 loop disruption and transphosphorylation are necessary for signalling. Thus, effector perturbation of one kinase molecule in the complex activates another. Hence, the Pto/Prf complex is a sophisticated molecular trap for effectors that target protein kinases, an essential aspect of the pathogen's virulence strategy. The data presented here give a clear view of why bacterial virulence and host recognition mechanisms are so often related and how the slowly evolving host is able to keep pace with the faster-evolving pathogen.

Figure 1. Phosphorylation of Pto upon activation of signalling.

(A) Slow migration of Prf-associated Pto after effector recognition. The indicated Prf, AvrPto and AvrPtoB constructs were transiently expressed in stable transgenic 35S:Pto N. benthamiana plants. Three days post infiltration, Prf-3HA and prf^K1128A-3HA proteins were immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) for Prf and Pto were performed with the antibodies indicated on the left. (B) A functional Prf molecule is required to generate the slow-migrating Pto form. The indicated Prf constructs were transiently expressed in stable transgenic 35S:Pto N. benthamiana plants. Three days post infiltration, Prf-3HA, prf^D1416V-3HA and N-term-3HA (prf^1–546-3HA) proteins were immunoprecipitated using anti-HA antibodies. Immunoblots were performed with the antibodies indicated on the left. Equal protein loading was verified by Coomassie Brilliant Blue (CBB) staining of the membranes. The experiment was repeated six times and typical results are shown.

Figure 2. Phosphorylation on Pto residues S198 and T199 is required for signalling.

(A) Trypan blue staining of cell death in N. benthamiana leaves. Pto-FLAG, pto mutant-FLAG, AvrPto, AvrPtoB, and prf^D1416V-3HA constructs were transiently expressed in Pro_Prf:Prf-5Myc or wild-type N. benthamiana as indicated and the tissue was stained 2 days post infiltration. The bar indicates 0.5 mm. Dead cells stain dark blue in this qualitative assay. Each row is derived from a single leaf, within which relative amounts of cell death were comparable, and is representative of six replicates. (B) The slow-migrating form of Pto requires kinase activity and double phosphorylation. Pto-FLAG, pto mutant-FLAG, AvrPto, and Prf-3HA constructs were transiently expressed in wild-type N. benthamiana as indicated, Prf-3HA was immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) were performed with the antibodies indicated on the left. (C) Quantification of the relative abundance of slow- and fast-migrating forms of Pto under elicitation conditions as described in B with Quantity One, Bio-Rad (adjusted volume = [CNT*mm2] data counts/mm²). Error bars are standard deviation of relative abundance between the same samples in independent immunoblots, probed with anti-Pto antibody.

Figure 3. Transphosphorylation is required for Ser-198 and Thr-199 phosphorylation.

(A) In trans inhibition of Pto S198 and T199 phosphorylation. The slower migrating from of Pto is suppressed in trans by prf^K1128A and pto^D164N, but not pto^S189A/T199A, pto^S189A and pto^T199A. Pto-5Myc, Pto-FLAG, pto mutant-FLAG, AvrPto, Prf-3HA, prf^K1128A-3HA constructs were transiently expressed in wild-type N. benthamiana as indicated, Prf-3HA and prf^K1128A-3HA were immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) were performed with the antibodies indicated on the left. (B) The slower migrating from of Pto was suppressed in trans by prf^K1128A and pto^D164N, but not by pto^S189A pto^T199A and pto^S189A/T199A. Pto-5Myc, Pto-FLAG, pto mutant-FLAG, AvrPto, Prf-3HA, prf^K1128A-3HA constructs were transiently expressed in wild-type N. benthamiana as indicated. Prf-3HA and prf^K1128A-3HA were immunoprecipitated using anti-HA antibodies. The relative abundance of slow- and fast-migrating forms of Pto-5Myc after AvrPto recognition was quantified two days post infiltration, from anti-Myc immunoblots using Quantity One, Bio-Rad (adjusted volume = [CNT*mm2] data counts/mm²).

Figure 4. Transphosphorylation is required for signalling.

(A) The phospho-mimic mutant pto^S198D/T199D induced cell death after AvrPto and AvrPtoB recognition. pto^S198D/T199D -FLAG, pto^{D164N/S198D/T199D}-FLAG, AvrPto, and AvrPtoB constructs were transiently expressed in Pro_Prf:Prf-5Myc N. benthamiana as indicated. Cell death was visualized with trypan blue staining two days post infiltration. Relative accumulation of pto^S198D/T199D and pto^{D164N/S198D/T199D} was detected with immunoblot (IB) with aντι-Pto antibody. Coomassie Brilliant Blue (CBB) staining of the IB membrane verified equal protein loading. (B) AvrPto-induced signalling by the phospho-mimic mutant pto^S198D/T199D is not suppressed in trans by pto^D164N. Pto-FLAG, pto^S198D/T199D-FLAG, pto^D164N-HA and AvrPto constructs were transiently expressed in Pro_Prf:Prf-5Myc N. benthamiana as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with the indicated antibodies. CBB staining of the IB membranes verified equal protein loading. (C) Phosphorylation of the kinase-inactive, constitutive gain-of-function (CGF) mutant pto^L205D at Ser-198 and Thr-199 is required for its hypersensitive cell death response inducing ability. pto^L205D-FLAG, pto^{S198A/T199A/L205D}-FLAG and pto^S198A/T199A-FLAG constructs were transiently expressed in Pro_Prf:Prf-5Myc N. benthamiana as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with anti-Pto antibody. CBB staining of the IB membranes verified equal protein loading. (D) Signalling by the kinase-inactive, CGF mutant pto^L205D mutant is suppressed in trans by pto^D164N. pto^L205D-HA, pto^D164N-FLAG, pto^S198D/T199D-FLAG, pto^S198A/T199A-FLAG and Pto-FLAG constructs were transiently expressed in Pro_Prf:Prf-5Myc N. benthamiana as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with the indicated antibodies. CBB staining of the IB membranes verified equal protein loading. For all images, the bar indicates 0.5 mm. Each row of trypan blue staining is derived from a single leaf, within which relative amounts of cell death were comparable, and representative of six replicates.

Figure 5. Model of sensor and helper kinases.

Dimerization of the NB-LRR protein Prf bring into proximity two molecules of Pto kinase (sensor and helper). Binding of AvrPto/AvrPtoB to previously phosphorylated Pto-sensor molecule disrupts the P+1 loop and hence negative regulation imposed by Prf. Derepression of the P+1 loop activates a second Pto-helper molecule in the Prf complex that transphosphorylates the first sensor-kinase, leading to complex activation.