RESUMO
Many plant species monitor and respond to changes in day length (photoperiod) for aligning reproduction with a favourable season. Day length is measured in leaves and, when appropriate, leads to the production of floral stimuli called florigens that are transmitted to the shoot apical meristem to initiate inflorescence development1. Rice possesses two florigens encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1)2. Here we show that the arrival of Hd3a and RFT1 at the shoot apical meristem activates FLOWERING LOCUS T-LIKE 1 (FT-L1), encoding a florigen-like protein that shows features partially differentiating it from typical florigens. FT-L1 potentiates the effects of Hd3a and RFT1 during the conversion of the vegetative meristem into an inflorescence meristem and organizes panicle branching by imposing increasing determinacy to distal meristems. A module comprising Hd3a, RFT1 and FT-L1 thus enables the initiation and balanced progression of panicle development towards determinacy.
Assuntos
Florígeno , Oryza , Florígeno/metabolismo , Meristema/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Flores , Reprodução , Regulação da Expressão Gênica de Plantas , Oryza/metabolismoRESUMO
The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.
Assuntos
Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Potexvirus/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Solanum tuberosum/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Imunológicos/genética , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/virologiaRESUMO
Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.
Assuntos
Resistência à Doença/genética , Mutação , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/genética , Solanaceae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Morte Celular , Cladosporium/metabolismo , Cladosporium/patogenicidade , Genes de Plantas , Leucina/metabolismo , Proteínas de Repetições Ricas em Leucina , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Estrutura Molecular , Mutagênese , Nucleotídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Solanaceae/metabolismo , Solanaceae/microbiologia , Nicotiana/genética , Nicotiana/microbiologia , Verticillium/metabolismo , Verticillium/patogenicidadeRESUMO
Hydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins. We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity. ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs. In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse.
Assuntos
Plântula/enzimologia , Plântula/imunologia , Serina Proteases/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/imunologia , Cladosporium/fisiologia , Espaço Extracelular/metabolismo , Umidade , Espaço Intracelular/metabolismo , Solanum lycopersicum/microbiologia , Plântula/microbiologia , TemperaturaRESUMO
The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.
Assuntos
Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Cladosporium/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Immunoblotting , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Glicoproteínas de Membrana/genética , Microscopia Confocal , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Interferência de RNA , Receptores de Superfície Celular/genética , Verticillium/fisiologiaRESUMO
Recently, we provided the first genetic evidence for the requirement of tomato PLC4 and PLC6 genes in defense activation and disease resistance. The encoded enzymes were catalytically active as they were able to degrade phosphatidylinositol (PI), thereby producing diacylglycerol (DG). Here we report differential PLC gene expression following the initiation of defense signaling by the interaction between Cladosporium fulvum resistance (R) protein Cf-4 and its matching effector Avr4 in tomato hybrid seedlings that express both Cf-4 and Avr4. Furthermore, we observed that PLC3 and PLC6 gene expression is upregulated by elevated temperature in the control seedlings. This upregulation coincides with an increase in the levels of phosphatidic acid (PA) and a decrease in the levels of PI and phosphatidylinositol phosphate (PIP). The decrease in PI and PIP levels matches with the activation of PLC. In addition, the levels of the structural phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined transiently during recovery after the exposure to elevated temperature., Further studies will be required to explain the mechanism causing the sustained accumulation of PA during recovery, combined with a reduction in the levels of structural phospholipids.
Assuntos
Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Ácidos Fosfatídicos/metabolismo , Doenças das Plantas/genética , Solanum lycopersicum/genética , Fosfolipases Tipo C/genética , Adaptação Fisiológica/genética , Cladosporium , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes de Plantas , Hibridização Genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/microbiologia , Glicoproteínas de Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plântula , Estresse Fisiológico/genética , Fosfolipases Tipo C/metabolismo , Regulação para CimaRESUMO
Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.
Assuntos
Resistência à Doença , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/biossíntese , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Sequência de Aminoácidos , Cladosporium/fisiologia , Inativação Gênica , Glicosilação , Proteínas de Fluorescência Verde/isolamento & purificação , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Transformação GenéticaRESUMO
Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum f. sp. lycopersici resistance protein of the CC-NB-LRR family, were identified. Sequence analysis revealed that I2I-1 belongs to the Formin gene family (SlFormin) whereas I2I-2 has homology to translin-associated protein X (SlTrax). SlFormin required only the N-terminal CC I-2 domain for binding, whereas SlTrax required both I-2 CC and part of the NB-ARC domain. Tomato plants stably silenced for these interactors were not compromised in I-2-mediated disease resistance. When extended or mutated forms of I-2 were used as baits, distinct and often opposite, interaction patterns with the two interactors were observed. These interaction patterns correlated with the proposed activation state of I-2 implying that active and inactive R proteins adopt distinct conformations. It is concluded that the yeast two hybrid system can be used as a proxy to monitor these different conformational states.
Assuntos
Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Solanum lycopersicum/metabolismo , Bioensaio , Fusarium/fisiologia , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Proteínas Mutantes/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
Nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors from plants confer resistance to fungal pathogens and many other pathogenic organisms. Their low expression makes it challenging to purify these receptors from plants in sufficient quantities to be able to identify interacting proteins by mass spectrometry. Here we describe a protocol to affinity-purify recombinant NB-LRR immune receptors, fused to the streptavidin-binding peptide tag.
Assuntos
Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas de Plantas/isolamento & purificação , Plantas/imunologia , Receptores Imunológicos/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Plantas/imunologia , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
The potato (Solanum tuberosum) nucleotide binding-leucine-rich repeat immune receptor Rx confers resistance to Potato virus X (PVX) and requires Ran GTPase-activating protein 2 (RanGAP2) for effective immune signaling. Although Rx does not contain a discernible nuclear localization signal, the protein localizes to both the cytoplasm and nucleus in Nicotiana benthamiana. Transient coexpression of Rx and cytoplasmically localized RanGAP2 sequesters Rx in the cytoplasm. This relocation of the immune receptor appeared to be mediated by the physical interaction between Rx and RanGAP2 and was independent of the concomitant increased GAP activity. Coexpression with RanGAP2 also potentiates Rx-mediated immune signaling, leading to a hypersensitive response (HR) and enhanced resistance to PVX. Besides sequestration, RanGAP2 also stabilizes Rx, a process that likely contributes to enhanced defense signaling. Strikingly, coexpression of Rx with the Rx-interacting WPP domain of RanGAP2 fused to a nuclear localization signal leads to hyperaccumulation of both the WPP domain and Rx in the nucleus. As a consequence, both Rx-mediated resistance to PVX and the HR induced by auto-active Rx mutants are significantly suppressed. These data show that a balanced nucleocytoplasmic partitioning of Rx is required for proper regulation of defense signaling. Furthermore, our data indicate that RanGAP2 regulates this partitioning by serving as a cytoplasmic retention factor for Rx.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Plantas/fisiologia , Solanaceae/fisiologia , Western Blotting , Proteínas de Plantas/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
The Rx1 protein, as many resistance proteins of the nucleotide binding-leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Plantas/metabolismo , Solanum/metabolismo , Frações Subcelulares/metabolismoRESUMO
Nucleotide binding leucine-rich repeat (NB-LRR) proteins play an important role in plant and mammalian innate immunity. In plants, these resistance proteins recognize specific pathogen-derived effector proteins. Recognition subsequently triggers a rapid and efficient defense response often associated with the hypersensitive response and other poorly understood processes that suppress the pathogen. To investigate mechanisms associated with the activation of disease resistance responses, we investigated proteins binding to the potato (Solanum tuberosum) NB-LRR protein Rx that confers extreme resistance to Potato virus X (PVX) in potato and Nicotiana benthamiana. By affinity purification experiments, we identified an endogenous N. benthamiana Ran GTPase-Activating Protein2 (RanGAP2) as an Rx-associated protein in vivo. Further characterization confirmed the specificity of this interaction and showed that the association occurs through their N-terminal domains. By specific virus-induced gene silencing of RanGAP2 in N. benthamiana carrying Rx, we demonstrated that this interaction is required for extreme resistance to PVX and suggest that RanGAP2 is part of the Rx signaling complex. These results implicate RanGAP-mediated cellular mechanisms, including nucleocytoplasmic trafficking, in the activation of disease resistance.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Nicotiana/virologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Potexvirus/fisiologia , Proteínas/metabolismo , Solanum tuberosum/virologia , Proteínas Ativadoras de GTPase/isolamento & purificação , Inativação Gênica , Imunidade Inata , Proteínas de Repetições Ricas em Leucina , Dados de Sequência Molecular , Proteínas de Plantas/isolamento & purificação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/isolamento & purificação , Replicação ViralRESUMO
Specificity of the plant innate immune system is often conferred by resistance (R) proteins. Most R proteins contain leucine-rich repeats (LRRs), a central nucleotide-binding site (NBS) and a variable amino-terminal domain. The LRRs are mainly involved in recognition, whereas the amino-terminal domain determines signalling specificity. The NBS forms part of a nucleotide binding (NB)-ARC domain that presumably functions as a molecular switch. The conserved nature of NB-ARC proteins makes it possible to map mutations of R protein residues onto the crystal structures of related NB-ARC proteins, providing hypotheses for the functional roles of these residues. A functional model emerges in which the LRRs control the molecular state of the NB-ARC domain. Pathogen recognition triggers nucleotide-dependent conformational changes that might induce oligomerisation, thereby providing a scaffold for activation of downstream signalling components.
Assuntos
Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Biologia Molecular , Conformação ProteicaRESUMO
Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP).
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Proteínas de Plantas/química , Solanum lycopersicum/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Hidrólise , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Mutação Puntual , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein I-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the I-2 complex, we performed yeast two-hybrid screens using the I-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an I-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between I-2 and PP5, and between I-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the I-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for I-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by I-2, showing that HSP90 is required for I-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Arabidopsis/metabolismo , Morte Celular , Fusarium , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Imunidade Inata/genética , Chaperonas Moleculares/metabolismo , Doenças das Plantas , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Vírus de Plantas , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.