Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 65
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
Sci Rep ; 9(1): 7042, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065034

RESUMO

During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5'-3' mRNA decapping is a late step in NMD-related mRNA degradation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Caulimovirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Virais/metabolismo , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Caulimovirus/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Tabaco/genética , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido
3.
PLoS One ; 14(3): e0213087, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840696

RESUMO

The split GFP technique is based on the auto-assembly of GFP when two polypeptides-GFP1-10 (residues 1-214; the detector) and GFP11 (residues 215-230; the tag)-both non-fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells displayed in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging.


Assuntos
Arabidopsis/virologia , Caulimovirus/patogenicidade , Proteínas de Fluorescência Verde/genética , Proteínas Virais/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Caulimovirus/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Mutagênese Sítio-Dirigida , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/virologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/metabolismo
4.
Plasmid ; 101: 1-9, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30465791

RESUMO

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 µg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.


Assuntos
Organismos Aquáticos/genética , Plasmídeos/metabolismo , Saccharomycetales/genética , Esporos Fúngicos/genética , Transformação Genética , Organismos Aquáticos/classificação , Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Cinamatos/farmacologia , Eletroporação/métodos , Temperatura Alta , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Plasmídeos/química , Regiões Promotoras Genéticas , Federação Russa , Saccharomycetales/classificação , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismo , Água do Mar/microbiologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/metabolismo
5.
Viruses ; 10(12)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30558257

RESUMO

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation.


Assuntos
Caulimovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Caulimovirus/genética , Núcleo Celular/química , Núcleo Celular/virologia , Citoplasma/química , Citoplasma/virologia , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Fases de Leitura Aberta , Doenças das Plantas/virologia , Fator de Iniciação 3 em Procariotos/genética , Interferência de RNA , Tabaco/citologia , Tabaco/virologia , Transativadores/genética , Proteínas Virais/genética
6.
Virology ; 523: 15-21, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30059841

RESUMO

Arabidopsis thaliana ecotype En-2 is resistant to several strains of Cauliflower mosaic virus (CaMV), including strain W260, but is susceptible to strain NY8153. Resistance in En-2 is conditioned by a single, semi-dominant gene called CAR1. We constructed several recombinant infectious clones between W260 and NY8153 and evaluated their capability to infect En-2. This analysis showed that the capacity of NY8153 to break resistance in En-2 was conditioned by mutations within the CaMV gene 1, a gene that encodes a protein dedicated to cell-to-cell movement (P1), and conversely, that P1 of W260 is responsible for eliciting the plant defense response. A previous study had shown that P6 of W260 was responsible for overcoming resistance in Arabidopsis ecotype Tsu-0 and that P6 of CaMV strain CM1841 was responsible for triggering resistance. The present study now shows that a second gene of CaMV is targeted by Arabidopsis for plant immunity.


Assuntos
Arabidopsis/genética , Caulimovirus/genética , Resistência à Doença/genética , Interações Hospedeiro-Patógeno , Imunidade Vegetal/genética , Proteínas Virais/genética , Arabidopsis/imunologia , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Caulimovirus/metabolismo , Caulimovirus/patogenicidade , Regulação da Expressão Gênica , Engenharia Genética , Genótipo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Recombinação Genética , Transdução de Sinais , Proteínas Virais/metabolismo
7.
Annu Rev Phytopathol ; 56: 89-110, 2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-29852091

RESUMO

The first bacterial and viral avirulence ( avr) genes were cloned in 1984. Although virus and bacterial avr genes were physically isolated in the same year, the questions associated with their characterization after discovery were very different, and these differences had a profound influence on the narrative of host-pathogen interactions for the past 30 years. Bacterial avr proteins were subsequently shown to suppress host defenses, leading to their reclassification as effectors, whereas research on viral avr proteins centered on their role in the viral infection cycle rather than their effect on host defenses. Recent studies that focus on the multifunctional nature of plant virus proteins have shown that some virus proteins are capable of suppression of the same host defenses as bacterial effectors. This is exemplified by the P6 protein of Cauliflower mosaic virus (CaMV), a multifunctional plant virus protein that facilitates several steps in the infection, including modulation of host defenses. This review highlights the modular structure and multifunctional nature of CaMV P6 and illustrates its similarities to other, well-established pathogen effectors.


Assuntos
Vírus de Plantas/genética , Proteínas Virais/genética , Caulimovirus/genética , Caulimovirus/metabolismo , Interações Hospedeiro-Patógeno , Vírus de Plantas/metabolismo , Proteínas Virais/metabolismo
8.
Virology ; 520: 103-110, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29843054

RESUMO

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation.


Assuntos
Caulimovirus/genética , Proteínas de Ligação a DNA/metabolismo , Doenças das Plantas/virologia , Interferência de RNA , Proteínas Virais/metabolismo , Agrobacterium/genética , Caulimovirus/metabolismo , Caulimovirus/patogenicidade , Núcleo Celular/virologia , Citoplasma/virologia , Proteínas de Ligação a DNA/genética , Inativação Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Folhas de Planta/virologia , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/virologia , Potexvirus/genética , RNA/química , RNA/genética , Tabaco/citologia , Tabaco/virologia , Proteínas Virais/genética
9.
Plant Mol Biol ; 96(1-2): 179-196, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29327227

RESUMO

KEY MESSAGE: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.


Assuntos
Armoracia/metabolismo , Armoracia/virologia , Caulimovirus/genética , Caulimovirus/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Armoracia/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/virologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia
10.
Bioengineered ; 9(1): 124-133, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28453384

RESUMO

Ulva prolifera is a green macroalgae with an extremely high growth rate that can accumulate biomass with considerable protein content. To set up an available seaweed expression system, a prior step is to isolate endogenous and strong constitutive promoters. For this reason, the full-length genomic actin1 gene from U. prolifera (Upactin1) was cloned and its 5' flanking sequence was obtained by genome walking. The Upactin1 open reading frame consisted of 1134 nucleotides encoding 377 amino acid residues. Besides 4 exons and 3 introns in the coding region, an extra leader intron was identified in the 5' untranslated region. According to quantitative GUS assays based on transient expression, the promoter activity of the Upactin1 5' flanking region was found to be several times higher than that of the widely-used cauliflower mosaic virus 35S (CaMV35S) in all tested species of Ulva. In addition, precise deletion of the leader intron led to a significant decrease of promoter strength in U. prolifera, and almost entire loss of strength in U. linza and U. pertusa. To our knowledge, this is the first report to prove function of a leader intron in algae. The 5' flanking region of Upactin1 was shown to be a much stronger promoter than the foreign CaMV35S, and its activity was highly dependent on the presence of the leader intron. We propose that the Upactin1 promoter could serve as an endogenous and strong constitutive element for genetic engineering of U. prolifera.


Assuntos
Actinas/genética , Proteínas de Algas/genética , Genoma de Planta , Regiões Promotoras Genéticas , Alga Marinha/genética , Ulva/genética , Região 5'-Flanqueadora , Actinas/metabolismo , Proteínas de Algas/metabolismo , Sequência de Bases , Caulimovirus/genética , Caulimovirus/metabolismo , Clonagem Molecular , Éxons , Expressão Gênica , Engenharia Genética , Íntrons , Fases de Leitura Aberta , Filogenia , Alga Marinha/classificação , Alga Marinha/crescimento & desenvolvimento , Alga Marinha/metabolismo , Ulva/classificação , Ulva/crescimento & desenvolvimento , Ulva/metabolismo
11.
PLoS One ; 12(12): e0189062, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29253877

RESUMO

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.


Assuntos
Brassica napus/virologia , Caulimovirus/metabolismo , Caulimovirus/patogenicidade , Transativadores/química , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Caulimovirus/ultraestrutura , Corpos de Inclusão Viral/metabolismo , Corpos de Inclusão Viral/ultraestrutura , Proteínas Mutantes/metabolismo , Fenótipo , Domínios Proteicos , Protoplastos/metabolismo , Transcrição Reversa/genética , Relação Estrutura-Atividade , Virulência , Replicação Viral
12.
Virus Res ; 230: 59-62, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28109755

RESUMO

Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions.


Assuntos
Agrobacterium tumefaciens/genética , Clonagem Molecular/métodos , DNA Complementar/genética , Engenharia Genética/métodos , Lycopersicon esculentum/virologia , Nepovirus/genética , Agrobacterium tumefaciens/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Chenopodium quinoa/virologia , Células Clonais , Cucumis sativus/virologia , DNA Complementar/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , Nepovirus/metabolismo , Regiões Promotoras Genéticas , RNA Catalítico/genética , RNA Catalítico/metabolismo , Tabaco/virologia
13.
BMC Res Notes ; 8: 365, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26289422

RESUMO

BACKGROUND: Non-photosynthetic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins are distributed in Brassicaceae plants. Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are highly expressed in leaves and stems, while Arabidopsis thaliana WSCP (AtWSCP) and Raphanus sativus WSCP (RshWSCP) are highly transcribed in floral organs. BoWSCP and LvWSCP exist in the endoplasmic reticulum (ER) body. However, the subcellular localization of AtWSCP and RshWSCP is still unclear. To determine the subcellular localization of these WSCPs, we constructed transgenic plants expressing Venus-fused AtWSCP or RshWSCP. RESULTS: Open reading frames corresponding to full-length AtWSCP and RshWSCP were cloned and ligated between the cauliflower mosaic virus 35S promoter and Venus, a gene encoding a yellow fluorescent protein. We introduced the constructs into A. thaliana by the floral dip method. We succeeded in constructing a number of transformants expressing Venus-fused chimeric AtWSCP (AtWSCP::Venus) or RshWSCP (RshWSCP::Venus). We detected fluorescence derived from the chimeric proteins using a fluorescence microscope system. In cotyledons, fluorescence derived from AtWSCP::Venus and RshWSCP::Venus was detected in spindle structures. The spindle structures altered their shape to a globular form under blue light excitation. In true leaves, the number of spindle structures was drastically reduced. These observations indicate that the spindle structure was the ER body. CONCLUSIONS: AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.


Assuntos
Arabidopsis/genética , Proteínas de Ligação à Clorofila/genética , Cotilédone/genética , Retículo Endoplasmático/genética , Regulação da Expressão Gênica de Plantas , Raphanus/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Clorofila/metabolismo , Proteínas de Ligação à Clorofila/metabolismo , Cotilédone/metabolismo , Cotilédone/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/ultraestrutura , Regiões Promotoras Genéticas , Ligação Proteica , Raphanus/metabolismo , Raphanus/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura
14.
Virology ; 476: 26-36, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25506670

RESUMO

Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1-D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection.


Assuntos
Caulimovirus/metabolismo , Corpos de Inclusão Viral/metabolismo , Doenças das Plantas/virologia , Tabaco/virologia , Transativadores/química , Transativadores/genética , Caulimovirus/química , Caulimovirus/genética , Caulimovirus/patogenicidade , Corpos de Inclusão Viral/genética , Mutação , Estrutura Terciária de Proteína , Transativadores/metabolismo , Virulência
15.
Plant Physiol ; 166(3): 1345-58, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25239023

RESUMO

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.


Assuntos
Proteínas de Arabidopsis/metabolismo , Caulimovirus/metabolismo , Plasmodesmos/metabolismo , Proteínas Virais/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caulimovirus/patogenicidade , Membrana Celular/metabolismo , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Corpos de Inclusão Viral/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Vírion/metabolismo
16.
Plant Physiol ; 164(3): 1261-70, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24477592

RESUMO

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) µA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.


Assuntos
Caulimovirus/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Vesículas Transportadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/virologia , Brassica rapa/efeitos dos fármacos , Brassica rapa/virologia , Brefeldina A/farmacologia , Caulimovirus/efeitos dos fármacos , Caulimovirus/patogenicidade , Compartimento Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas do Movimento Viral em Plantas/química , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Tabaco/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Tirosina/metabolismo , Tirfostinas/farmacologia
17.
Transgenic Res ; 23(2): 235-44, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24036909

RESUMO

Constitutive promoters are essential tools for analyses of gene functions by transgenic approaches. For overexpression and silencing studies of genes, a ubiquitous and strong expression of genes under investigation as well as selection markers is preferred. For future applications in the emerging basal plant model system Marchantia polymorpha, a liverwort, activities of the viral 35S cauliflower mosaic virus promoter and the endogenous elongation factor 1α (MpEF1α) promoter were analyzed. Expression of the reporter gene ß-glucuronidase (GUS), driven by the CaMV35 and MpEF1α promoters, was compared throughout plant development. Significant differences were observed between the two promoter activities. The CaMV35 promoter yields a weak reporter gene expression in the meristematic zones but drives a strong expression in the thallus. The MpEF1α promoter causes a strong meristematic GUS expression and is more active in female sexual tissues. Overall, the MpEF1α promoter seems to be the better option for obtaining a strong and ubiquitous transgene expression. Furthermore, a whole mount in situ hybridization protocol for Marchantia was established. Analysis of MpEF1α mRNA transcript in intact, whole tissues showed an expression pattern that is overall similar to the pattern of the GUS reporter gene expression driven by the MpEF1α promoter, including strong expression in meristematic zones. The whole mount technique reported here can be used to determine the mRNA expression in intact gemmae and archegonia, and has the potential to be applied for screening large numbers of transgenic plants, for instance to identify knock-down mutants.


Assuntos
Caulimovirus/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Transferência de Genes , Genes de Plantas/genética , Marchantia/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Caulimovirus/metabolismo , Primers do DNA , Perfilação da Expressão Gênica , Hibridização In Situ , Marchantia/genética , Meristema/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo
18.
Plant Signal Behav ; 9(7): e29121, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25763491

RESUMO

Plant virus genomes cross the barrier of the host cell wall and move to neighboring cells either in the form of nucleoprotein complex or encapsidated into virions. Virus transport is facilitated by virus-encoded movement proteins (MP), which are different from one another in number, size, sequence, and in the strategy used to overcome the size exclusion limit of plasmodesmata (PD). (1) A group of them forms tubules inside the lumen of highly modified PDs upon removal of the desmotubule. To date the molecular mechanism(s) and the host factors involved in the assembly of MP tubules as well as the mechanistic aspects of virus particle transport throughout them remain substantially unknown. In a recent study, we showed that Cauliflower mosaic virus (CaMV) MP traffics in the endocytic pathway with the help of 3 tyrosine-sorting signals, which are not required to target MP to the plasma membrane but are essential for tubule formation. (2) This evidence unravels a previously unknown connection between the plant endosomal system and tubule-mediated virus movement that is here supported by demonstration of hindrance of tubule assembly upon Brefeldin A (BFA) treatment. We discuss the implications of our data on the mechanisms of viral transport through tubules and draw parallels with plant mechanisms of polarized growth.


Assuntos
Brefeldina A/farmacologia , Caulimovirus/patogenicidade , Membrana Celular/metabolismo , Plasmodesmos/metabolismo , Tabaco/metabolismo , Proteínas Virais/metabolismo , Caulimovirus/metabolismo , Transporte Proteico , Tabaco/efeitos dos fármacos , Tabaco/virologia
19.
J Gen Virol ; 94(Pt 12): 2777-2789, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24088344

RESUMO

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.


Assuntos
Caulimovirus/patogenicidade , Estrutura Terciária de Proteína/genética , Interferência de RNA/efeitos dos fármacos , Ácido Salicílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores/genética , Transativadores/farmacologia , Sequência de Aminoácidos , Arabidopsis/virologia , Caulimovirus/genética , Caulimovirus/metabolismo , Dados de Sequência Molecular , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Deleção de Sequência , Transativadores/química , Transativadores/metabolismo , Replicação Viral
20.
Virology ; 443(2): 363-74, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23769239

RESUMO

The gene VI product, protein 6 (P6), of Cauliflower mosaic virus (CaMV) assembles into large, amorphous inclusion bodies (IBs) that are considered sites for viral protein synthesis and viral genome replication and encapsidation. P6 IBs align with microfilaments and require them for intracellular trafficking, a result implying that P6 IBs function to move virus complexes or virions within the cell to support virus physiology. Through a yeast two-hybrid screen we determined that CHUP1, a plant protein allowing chloroplast transport through an interaction with chloroplast and microfilament, interacts with P6. The interaction between CHUP1 and P6 was confirmed through colocalization in vivo and co-immunoprecipitation assays. A truncated CHUP1 fused with enhanced cyan fluorescent protein, unable to transport chloroplasts, inhibited intracellular movement of P6-Venus inclusions. Silencing of CHUP1 in N. edwardsonii impaired the ability of CaMV to infect plants. The findings suggest that CHUP1 supports CaMV infection through an interaction with P6.


Assuntos
Citoesqueleto de Actina/metabolismo , Caulimovirus/patogenicidade , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transativadores/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/virologia , Imunoprecipitação , Proteínas dos Microfilamentos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transativadores/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Virais/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...