RESUMO
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.
Assuntos
Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Solanum lycopersicum/metabolismo , Bactérias/patogenicidade , Proteínas de Bactérias , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Fosforilação , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Transdução de Sinais , VirulênciaRESUMO
Biotrophic 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 molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.
Assuntos
Arabidopsis/parasitologia , Oomicetos/genética , Doenças das Plantas/parasitologia , Arabidopsis/genética , Sequência de Bases , Evolução Biológica , Genes , Genoma , Interações Hospedeiro-Patógeno , Oomicetos/crescimento & desenvolvimento , Oomicetos/metabolismo , Simbiose/genéticaRESUMO
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.
Assuntos
Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Solanum lycopersicum/metabolismo , Sequência de Aminoácidos , Imunoprecipitação , Solanum lycopersicum/genética , Solanum lycopersicum/imunologia , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/químicaRESUMO
Tomato 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.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Mutação , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Pseudomonas syringae , Transdução de SinaisRESUMO
Plants 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.
Assuntos
Imunoprecipitação/métodos , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Centrifugação/métodos , Proteínas de Plantas/isolamento & purificaçãoRESUMO
Plant 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.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas syringae/patogenicidade , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Imunidade Inata , Solanum lycopersicum/genética , Proteínas Mutantes/metabolismo , Fosforilação , Doenças das Plantas/imunologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Pseudomonas syringae/genética , Pseudomonas syringae/crescimento & desenvolvimento , Pseudomonas syringae/metabolismo , Transdução de Sinais , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/metabolismoRESUMO
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.