RESUMO
DNA ß satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The ßC1 protein encoded by DNA ß is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The ßC1 proteins from various DNA ß molecules show low levels of amino acid sequence conservation. We show here that the ßC1 from DNA ß associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA ß satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which ßC1 protects a heterologous virus from host gene silencing.
Assuntos
Begomovirus/metabolismo , DNA Viral/metabolismo , Vírus Auxiliares/fisiologia , Doenças das Plantas/genética , Interferência de RNA , Vírus Satélites/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Begomovirus/genética , DNA Viral/genética , Vírus Auxiliares/genética , Doenças das Plantas/virologia , Vírus Satélites/genética , Nicotiana/genética , Nicotiana/virologia , Proteínas Virais/genéticaRESUMO
Tomato leaf curl virus-Australia (ToLCV) C4 protein has been shown to be associated with virus pathogenesis. Here, we demonstrate that C4 acts as a suppressor of gene silencing. To understand the multifunctional role of C4, a novel shaggy-like kinase (SlSK) from tomato, which interacts with ToLCV C4 in a yeast two-hybrid assay, was isolated and interaction between these proteins was confirmed in vitro and in planta. Using deletion analysis of C4, a 12 amino acid region in the C-terminal part of C4 was identified which was shown to be essential for its binding to SlSK. We further demonstrate that this region is not only important for the interaction of C4 with SlSK, but is also required for C4 function to suppress gene silencing activity and to induce virus symptoms in a PVX system. The potential significance of ToLCV C4 and SlSK interaction is discussed.
Assuntos
Begomovirus/patogenicidade , Geminiviridae/patogenicidade , Doenças das Plantas/virologia , Solanum lycopersicum/genética , Begomovirus/genética , Replicação do DNA , DNA Viral/genética , Inativação Gênica , Solanum lycopersicum/enzimologia , Solanum lycopersicum/virologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.
Assuntos
Geminiviridae/patogenicidade , Proteínas de Plantas/metabolismo , Solanum lycopersicum/virologia , Proteínas Virais/metabolismo , DNA Viral/metabolismo , Geminiviridae/genética , Geminiviridae/metabolismo , Biblioteca Gênica , Glicosilação , Proteínas de Fluorescência Verde/análise , Solanum lycopersicum/metabolismo , Cebolas/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/análise , Análise de Sequência de Proteína , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/fisiologiaRESUMO
Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.
Assuntos
Proteínas de Plantas/metabolismo , Vírus de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Viral/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Regulação Viral da Expressão Gênica/genética , Solanum lycopersicum/genética , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Vírus de Plantas/genética , Estrutura Terciária de Proteína/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Ativação Transcricional/genética , Regulação para Cima/genética , Proteínas Virais/genéticaRESUMO
We have previously identified an upstream 556-bp enhancer domain for the chicken CYP2H1 gene that responds to phenobarbital and binds several transcription factors, including the orphan chicken xenobiotic receptor (CXR). By contrast, the promoter lacks a CXR site and is not inducible by phenobarbital. Although it has been established that CXR can interact with the coactivator SRC-1, there are no reports as to whether other coactivators may be important for phenobarbital-mediated inducibility. Our studies using the adenovirus E1A wild-type protein, which inhibits the coactivators cAMP response element binding protein (CBP) and CBP associated factor (p/CAF), provide evidence for the involvement of one or both of these coactivators at the enhancer but not at the promoter of the CYP2H1 gene. The observations that mutant E1A proteins did not affect the enhancer activity and that inhibition by wild-type E1A was reversed by CBP and p/CAF confirmed the involvement of these coactivators in the induction process. We propose that the intrinsic histone acetyl transferase activity of one or both of these coactivators participates in chromatin remodeling thereby stimulating drug induction of the promoter. This proposal was supported by experiments with the histone deacetylase inhibitor, trichostatin A, which resulted in the superinduction of the drug response but had little effect on basal expression of the CYP2H1 gene. The work provides evidence for the first time for the involvement of the coactivators CBP and p/CAF in the phenobarbital-mediated induction of the CYP2H1 gene.
