20 research outputs found
Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana
No full textThe Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf
The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation
No full textThe 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
Identification of post-translational modifications of plant protein complexes
No full textPlants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein
Regulation of Tomato Prf by Pto-like Protein Kinases
No full textTomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N- terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors
Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana
No full textBiotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa.
From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of
molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires ‘‘effectors’’ to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the ‘‘CHXCs’’, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterinrequiring
biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to
human pathogens within the Chromalveolata
Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition
No full textCytoplasmic 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
Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition
No full textCytoplasmic 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
Phosphorylation of Pto upon activation of signalling.
No full text<p>(<b>A</b>) Slow migration of Prf-associated Pto after effector recognition. The indicated Prf, AvrPto and AvrPtoB constructs were transiently expressed in stable transgenic 35S:<i>Pto N. benthamiana</i> plants. Three days post infiltration, Prf-3HA and prf<sup>K1128A</sup>-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>B</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:<i>Pto N. benthamiana</i> plants. Three days post infiltration, Prf-3HA, prf<sup>D1416V</sup>-3HA and N-term-3HA (prf<sup>1–546</sup>-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.</p
Transphosphorylation is required for signalling.
No full text<p>(<b>A</b>) The phospho-mimic mutant pto<sup>S198D/T199D</sup> induced cell death after AvrPto and AvrPtoB recognition. pto<sup>S198D/T199D</sup> -FLAG, pto<sup>D164N/S198D/T199D</sup>-FLAG, AvrPto, and AvrPtoB constructs were transiently expressed in Pro<sub>Prf</sub>:<i>Prf-5Myc N. benthamiana</i> as indicated. Cell death was visualized with trypan blue staining two days post infiltration. Relative accumulation of pto<sup>S198D/T199D</sup> and pto<sup>D164N/S198D/T199D</sup> 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<sup>S198D/T199D</sup> is not suppressed <i>in trans</i> by pto<sup>D164N</sup>. Pto-FLAG, pto<sup>S198D/T199D</sup>-FLAG, pto<sup>D164N</sup>-HA and AvrPto constructs were transiently expressed in Pro<sub>Prf</sub>:<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<sup>L205D</sup> at Ser-198 and Thr-199 is required for its hypersensitive cell death response inducing ability. pto<sup>L205D</sup>-FLAG, pto<sup>S198A/T199A/L205D</sup>-FLAG and pto<sup>S198A/T199A</sup>-FLAG constructs were transiently expressed in Pro<sub>Prf</sub>:<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<sup>L205D</sup> mutant is suppressed <i>in trans</i> by pto<sup>D164N</sup>. pto<sup>L205D</sup>-HA, pto<sup>D164N</sup>-FLAG, pto<sup>S198D/T199D</sup>-FLAG, pto<sup>S198A/T199A</sup>-FLAG and Pto-FLAG constructs were transiently expressed in Pro<sub>Prf</sub>:<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
Host inhibition of a bacterial virulence effector triggers immunity to infection
No full textPlant pathogenic bacteria secrete effector proteins that attack the host signaling machinery to suppress immunity. Effectors can be recognized by hosts leading to immunity. One such effector is AvrPtoB of Pseudomonas syringae, which degrades host protein kinases, such as tomato Fen, through an E3 ligase domain. Pto kinase, which is highly related to Fen, recognizes AvrPtoB in conjunction with the resistance protein Prf. Here we show that Pto is resistant to AvrPtoB-mediated degradation because it inactivates the E3 ligase domain. AvrPtoB ubiquitinated Fen within the catalytic cleft, leading to its breakdown and loss of the associated Prf protein. Pto avoids this by phosphorylating and inactivating the AvrPtoB E3 domain. Thus, inactivation of a pathogen virulence molecule is one mechanism by which plants resist disease
