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

The bacteria Pseudomonas syringae is a pathogen of many crop species and one of the model pathogens for studying plant and bacterial arms race coevolution. In the current model, plants perceive bacteria pathogens via plasma membrane receptors, and recognition leads to the activation of general defenses. In turn, bacteria inject proteins called effectors into the plant cell to prevent the activation of immune responses. AvrPto and AvrPtoB are two such proteins that inhibit multiple plant kinases. The tomato plant has reacted to these effectors by the evolution of a cytoplasmic resistance complex. This complex is compromised of two proteins, Prf and Pto kinase, and is capable of recognizing the effector proteins. How the Pto kinase is able to avoid inhibition by the effector proteins is currently unknown. Our data shows how the tomato plant utilizes dimerization of resistance proteins to gain advantage over the faster evolving bacterial pathogen. Here we illustrate that oligomerisation of Prf brings into proximity two Pto kinases allowing them to avoid inhibition by the effectors by transphosphorylation and to activate immune responses

Phevamine A, a small molecule that suppresses plant immune responses

Bacterial plant pathogens cause significant crop damage worldwide. They invade plant cells by producing a variety of virulence factors, including small-molecule toxins and phytohormone mimics. Virulence of the model pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) is regulated in part by the sigma factor HrpL. Our study of the HrpL regulon identified an uncharacterized, three-gene operon in Pto that is controlled by HrpL and related to the Erwinia hrp-associated systemic virulence (hsv) operon. Here, we demonstrate that the hsv operon contributes to the virulence of Pto on Arabidopsis thaliana and suppresses bacteria-induced immune responses. We show that the hsv-encoded enzymes in Pto synthesize a small molecule, phevamine A. This molecule consists of L-phenylalanine, L-valine, and a modified spermidine, and is different from known small molecules produced by phytopathogens. We show that phevamine A suppresses a potentiation effect of spermidine and L-arginine on the reactive oxygen species burst generated upon recognition of bacterial flagellin. The hsv operon is found in the genomes of divergent bacterial genera, including ∼37% of P. syringae genomes, suggesting that phevamine A is a widely distributed virulence factor in phytopathogens. Our work identifies a small-molecule virulence factor and reveals a mechanism by which bacterial pathogens overcome plant defense. This work highlights the power of omics approaches in identifying important small molecules in bacteria–host interactions

Phevamine A, a small molecule that suppresses plant immune responses

Bacterial plant pathogens cause significant crop damage worldwide. They invade plant cells by producing a variety of virulence factors, including small-molecule toxins and phytohormone mimics. Virulence of the model pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) is regulated in part by the sigma factor HrpL. Our study of the HrpL regulon identified an uncharacterized, three-gene operon in Pto that is controlled by HrpL and related to the Erwinia hrp-associated systemic virulence (hsv) operon. Here, we demonstrate that the hsv operon contributes to the virulence of Pto on Arabidopsis thaliana and suppresses bacteria-induced immune responses. We show that the hsv-encoded enzymes in Pto synthesize a small molecule, phevamine A. This molecule consists of L-phenylalanine, L-valine, and a modified spermidine, and is different from known small molecules produced by phytopathogens. We show that phevamine A suppresses a potentiation effect of spermidine and L-arginine on the reactive oxygen species burst generated upon recognition of bacterial flagellin. The hsv operon is found in the genomes of divergent bacterial genera, including ∼37% of P. syringae genomes, suggesting that phevamine A is a widely distributed virulence factor in phytopathogens. Our work identifies a small-molecule virulence factor and reveals a mechanism by which bacterial pathogens overcome plant defense. This work highlights the power of omics approaches in identifying important small molecules in bacteria–host interactions

Functional characterisation of tomato Prf during signal transduction by Pto

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Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition

Cytoplasmic recognition of pathogen virulence effectors by plant NB-LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB-LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N-terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N-terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non-Pto kinases into the Prf complex extends the number of effector proteins that can be recognized

The Tomato NBARC-LRR Protein Prf Interacts with Pto Kinase in Vivo to Regulate Specific Plant Immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch

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

<p>(<b>A</b>) Trypan blue staining of cell death in <i>N. benthamiana</i> leaves. Pto-FLAG, pto mutant-FLAG, AvrPto, AvrPtoB, and prf^D1416V-3HA constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc</i> or wild-type <i>N. benthamiana</i> 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>B</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 <i>N. benthamiana</i> as indicated, Prf-3HA was immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) were performed with the antibodies indicated on the left. (<b>C</b>) 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.</p

Transphosphorylation is required for signalling.

<p>(<b>A</b>) 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:<i>Prf-5Myc N. benthamiana</i> 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>B</b>) AvrPto-induced signalling by the phospho-mimic mutant pto^S198D/T199D is not suppressed <i>in trans</i> by pto^D164N. Pto-FLAG, pto^S198D/T199D-FLAG, pto^D164N-HA and AvrPto constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc N. benthamiana</i> 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. (<b>C</b>) 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:<i>Prf-5Myc N. benthamiana</i> 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. (<b>D</b>) Signalling by the kinase-inactive, CGF mutant pto^L205D mutant is suppressed <i>in trans</i> 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:<i>Prf-5Myc N. benthamiana</i> 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.</p

Summary of Pto signalling activities.

1<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat.1003123.s006" target="_blank">Figure S6</a>.</p>2<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat-1003123-g002" target="_blank">Figure 2A</a>.</p>3<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat.1003123.s007" target="_blank">Figure S7</a>.</p