RESUMO
LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box-dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant-microbe interactions and development through interacting with receptor-like kinases is discussed.
Assuntos
Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Ubiquitina-Proteína Ligases/metabolismo , Regulação da Expressão Gênica de Plantas , Medicago truncatula/enzimologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Interferência de RNA , Transdução de Sinais , Sinorhizobium meliloti/fisiologia , Simbiose/genética , Nicotiana/enzimologia , Nicotiana/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genéticaRESUMO
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
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 Arabidopsis transcription factor AtMYB30 was previously identified as a positive regulator of plant hypersensitive cell death and defence responses to inoculation with bacterial pathogens. In this study, we attempted to generate Arabidopsis transgenic lines that overexpress AtMYB30 under the control of the constitutive 35S promoter. However, no transgenic lines overexpressing AtMYB30 could be obtained, suggesting the existence of a molecular mechanism that negatively regulates AtMYB30 expression in planta. Our results suggest that RNA silencing directly mediates downregulation of AtMYB30 expression, both in young seedlings and in adult plants. In contrast, an indirect RNA silencing mechanism is responsible for the induction of AtMYB30 expression after bacterial inoculation, possibly via the degradation of a yet unknown negative regulator of its expression. These results underline the importance of RNA silencing in the regulation of the activity of transcription factors both during plant development and in response to microbes.