RESUMO
Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid-binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant vad1-1 has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, associated with defense gene expression and increased resistance to Pseudomonas strains. To test the potential of ectopic expression of VAD1 to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure-function analysis of VAD1 by transient over-expression in Nicotiana benthamiana and by complementation of the mutant vad1-1. We found that (i) overexpression of VAD1 controls negatively the HR cell death and defense expression either transiently in Nicotiana benthamania or in Arabidopsis plants in response to avirulent strains of Pseudomonas syringae, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/imunologia , Morte Celular/imunologia , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/imunologia , Morte Celular/genética , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Núcleo Celular/microbiologia , Citosol/imunologia , Citosol/metabolismo , Citosol/microbiologia , Teste de Complementação Genética , Mutação , Células Vegetais/imunologia , Células Vegetais/metabolismo , Células Vegetais/microbiologia , Doenças das Plantas/genética , Domínios Proteicos , Pseudomonas syringae/crescimento & desenvolvimento , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
One of the most efficient plant resistance reactions to pathogen attack is the hypersensitive response, a form of programmed cell death at infection sites. The Arabidopsis transcription factor MYB30 is a positive regulator of hypersensitive cell death responses. Here we show that MIEL1 (MYB30-Interacting E3 Ligase1), an Arabidopsis RING-type E3 ubiquitin ligase that interacts with and ubiquitinates MYB30, leads to MYB30 proteasomal degradation and downregulation of its transcriptional activity. In non-infected plants, MIEL1 attenuates cell death and defence through degradation of MYB30. Following bacterial inoculation, repression of MIEL1 expression removes this negative regulation allowing sufficient MYB30 accumulation in the inoculated zone to trigger the hypersensitive response and restrict pathogen growth. Our work underlines the important role played by ubiquitination to control the hypersensitive response and highlights the sophisticated fine-tuning of plant responses to pathogen attack. Overall, this work emphasizes the importance of protein modification by ubiquitination during the regulation of transcriptional responses to stress in eukaryotic cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica de Plantas , Microscopia Confocal , Células Vegetais/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Pseudomonas syringae/fisiologia , Nicotiana/genética , Fatores de Transcrição/genética , Transcrição Gênica , UbiquitinaçãoRESUMO
The hypersensitive response (HR), characterized by a rapid and localized cell death at the inoculation site, is one of the most efficient resistance reactions to pathogen attack in plants. The transcription factor AtMYB30 was identified as a positive regulator of the HR and resistance responses during interactions between Arabidopsis and bacteria. Here, we show that AtMYB30 and the secreted phospholipase AtsPLA(2)-alpha physically interact in vivo, following the AtMYB30-mediated specific relocalization of AtsPLA(2)-alpha from cytoplasmic vesicles to the plant cell nucleus. This protein interaction leads to repression of AtMYB30 transcriptional activity and negative regulation of plant HR. Moreover, Atspla(2)-alpha mutant plants are more resistant to bacterial inoculation, whereas AtsPLA(2)-alpha overexpression leads to decreased resistance, confirming that AtsPLA(2)-alpha is a negative regulator of AtMYB30-mediated defense. These data underline the importance of cellular dynamics and, particularly, protein translocation to the nucleus, for defense-associated gene regulation in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fosfolipases A2 Secretórias/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Sequência de Bases , Primers do DNA/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Mutação , Fosfolipases A2 Secretórias/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/patogenicidade , RNA de Plantas/genética , RNA de Plantas/metabolismo , Nicotiana/genética , Nicotiana/microbiologia , Nicotiana/fisiologia , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD(1-760)) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD(216-760)). Furthermore, the N-terminal extension of XopD, which is absent in XopD(216-760), is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.
Assuntos
Xanthomonas campestris/metabolismo , Sequência de Aminoácidos , DNA/química , Proteínas de Ligação a DNA/química , Dimerização , Epitopos/química , Escherichia coli/metabolismo , Teste de Complementação Genética , Solanum lycopersicum/microbiologia , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/microbiologiaRESUMO
Although ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/citologia , Arabidopsis/genética , Morte Celular/fisiologia , Etilenos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Mutação , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Transdução de SinaisRESUMO
The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Canais Iônicos/genética , Pseudomonas/crescimento & desenvolvimento , Alelos , Sequência de Aminoácidos , Animais , Apoptose/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Feminino , Regulação da Expressão Gênica de Plantas , Imunidade Inata/genética , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Mutação , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Potássio/metabolismo , Transdução de Sinais/genética , Sódio/metabolismo , XenopusRESUMO
Hypersensitive response (HR) is a programmed cell death that is commonly associated with disease resistance in plants. Among the different HR-related early induced genes, the AtMYB30 gene is specifically, rapidly, and transiently expressed during incompatible interactions between Arabidopsis and bacterial pathogens. Its expression was also shown to be deregulated in Arabidopsis mutants affected in the control of cell death initiation. Here, we demonstrate that overexpression in Arabidopsis and tobacco of AtMYB30 (i) accelerates and intensifies the appearance of the HR in response to different avirulent bacterial pathogens, (ii) causes HR-like responses to virulent strains, and (iii) increases resistance against different bacterial pathogens, and a virulent biotrophic fungal pathogen, Cercospora nicotianae. In antisense AtMYB30 Arabidopsis lines, HR cell death is strongly decreased or suppressed in response to avirulent bacterial strains, resistance against different bacterial pathogens decreased, and the expression of HR- and defense-related genes was altered. Taken together, these results strongly suggest that AtMYB30 is a positive regulator of hypersensitive cell death.