RESUMO
Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.
Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Animais , Humanos , Camundongos , Células Clonais , Leucemia Mieloide Aguda/genética , Mutação , Fenótipo , Proteína Supressora de Tumor p53/genética , Proteínas Mutantes , Dobramento de ProteínaRESUMO
Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10-56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to â¼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.
RESUMO
The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.
Assuntos
Hematopoiese Clonal/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Leucemia Mieloide/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Afidicolina/farmacologia , Calreticulina/genética , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Células Progenitoras Mieloides , Proteínas Repressoras/genética , Deleção de Sequência/genética , Fatores de Processamento de Serina-Arginina/genética , DNA Polimerase tetaRESUMO
Novel targeted therapies demonstrate improved survival in specific subgroups (defined by genetic variants) of acute myeloid leukemia (AML) patients, validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in high-throughput in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells (LSCs). Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in LSCs. We performed ex vivo sensitivity screening to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified group with sensitivity to several tyrosine kinase inhibitors (TKIs), including the multi-TKI, dasatinib, and searched for correlations between dasatinib response, exome sequencing and gene expression from our dataset and from the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (AUC=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibits LSC engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, LSCs from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Animais , Dasatinibe/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Transcriptoma , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Pancreatic ß-cell membranes and presynaptic areas of neurons contain analogous protein complexes that control the secretion of bioactive molecules. These complexes include the neuroligins (NLs) and their binding partners, the neurexins (NXs). It has been recently reported that both insulin secretion and the proliferation rates of ß-cells increase when cells are co-cultured with full-length NL-2 clusters. The pharmacological use of full-length protein is always problematic due to its unfavorable pharmacokinetic properties. Thus, NL-2-derived short peptide was conjugated to the surface of polyamidoamine-based (PAMAM) dendrimers. This nanoscale composite improved ß-cell functions in terms of the rate of proliferation, glucose-stimulated insulin secretion (GSIS), and functional maturation. This functionalized dendrimer also protected ß-cells under cellular stress conditions. In addition, various novel peptidomimetic scaffolds of NL-2-derived peptide were designed, synthesized, and conjugated to the surface of PAMAM in order to increase the biostability of the conjugates. However, after being covered by peptidomimetics, PAMAM dendrimers were inactive. Thus, the original peptide-based PAMAM dendrimer is a leading compound for continued research that might provide a unique starting point for designing an innovative class of antidiabetic therapeutics that possess a unique mode of action.
RESUMO
Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.
Assuntos
Antígenos CD34/biossíntese , Linhagem Celular Tumoral , Deleção Cromossômica , Inversão Cromossômica , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 7/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda , Proteínas de Neoplasias/biossíntese , Adulto , Antígenos CD34/genética , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Cromossomos Humanos Par 3/metabolismo , Cromossomos Humanos Par 7/metabolismo , Técnicas de Cocultura , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
CueR (Cu export regulator) is a metalloregulator protein that "senses" Cu(I) ions with very high affinity, thereby stimulating DNA binding and the transcription activation of two other metalloregulator proteins. The crystal structures of CueR when unbound or bound to DNA and a metal ion are very similar to each other, and the role of CueR and Cu(I) in initiating the transcription has not been fully understood yet. Using double electron-electron resonance (DEER) measurements and structure modeling, we investigate the conformational changes that CueR undergoes upon binding Cu(I) and DNA in solution. We observe three distinct conformations, corresponding to apo-CueR, DNA-bound CueR in the absence of Cu(I) (the "repression" state), and CueR-Cu(I)-DNA (the "activation" state). We propose a detailed structural mechanism underlying CueR's regulation of the transcription process. The mechanism explicitly shows the dependence of CueR activity on copper, thereby revealing the important negative feedback mechanism essential for regulating the intracellular copper concentration.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Ativação Transcricional , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cobre/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Ligação ProteicaRESUMO
Bacterial cells have developed sophisticated systems to deal with the toxicity of metal ions. Escherichia coli CusCFBA is a complex efflux system, responsible for transferring Cu(I) and Ag(I) ions; this system, located in the periplasm, involves four proteins, CusA, CusB, CusC, and CusF. CusA, CusB, and CusC are connected to one another in an oligomerization ratio of 3:6:3 CusA/CusB/CusC to form the CusCBA periplasm membrane transporter. CusB is an adaptor protein that connects the two membrane proteins CusA (inner membrane) and CusC (outer membrane). CusF is a metallochaperone that transfers Cu(I) and Ag(I) to the CusCBA transporter from the periplasm. The crystal structures of CusB, CusC, CusF, and the CusBA complex have been resolved, shedding some light on the efflux mechanism underlying this intriguing system. However, since CusB is an adaptor protein, its role in operating this system is significant, and should be understood in detail. Here, we utilize EPR spectroscopy to target the conformational changes that take place in the full CusB protein upon binding Cu(I). We reveal that CusB is a dimer in solution, and that the orientation of one molecule with respect to the other molecule changes upon Cu(I) coordination, resulting in a more compact CusB structure. These structural and topological changes upon Cu(I) binding probably play the role of a switch for opening the channel and transferring metal ions from CusB to CusC and out of the cell.
Assuntos
Cobre/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Dicroísmo Circular , Cobre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Proteínas de Escherichia coli/genética , Fusão de Membrana , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , SoluçõesRESUMO
C. elegans is a heme auxotroph that requires environmental heme for sustenance. As such, worms utilize HRG-3, a small heme-trafficking protein, to traffic heme from the intestine to extra-intestinal tissues and embryos. However, how HRG-3 binds and delivers heme remains unknown. In this study, we utilized electron paramagnetic resonance spectroscopy together with site-directed spin labeling, absorption spectroscopy, circular dichroism, and mutagenesis to gain structural and molecular insights into HRG-3. We showed that HRG-3 is a dimer, whereas H9 and H10 are significant residues that preserve a specific conformational state in the HRG-3 dimer. In the absence of H9 and H10, HRG-3 can still bind heme, although with a different affinity. Furthermore, the heme-binding site is closer to the N-termini than to the C-termini. Taken together, our results lay the groundwork for future mechanistic and structural studies of HRG-3 and inter-tissue heme trafficking in metazoans.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Hemeproteínas/química , Hemeproteínas/metabolismo , Histidina/química , Histidina/metabolismo , Animais , Caenorhabditis elegans/química , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Eletroforese em Gel de PoliacrilamidaRESUMO
The search for novel anticancer therapeutic agents is an urgent and important issue in medicinal chemistry. Here, we report on the biological activity of the copper-based bioinorganic complex Cu4 (2,4-di-tert-butyl-6-(1H-imidazo- [1, 10] phenanthrolin-2-yl)phenol)4]·10 CH3CN (2), which was tested in rat L6 myotubes, mouse NSC-34 motor neurone-like cells, and HepG-2 human liver carcinoma. Upon 96 h incubation, 2 exhibited a significant cytotoxic effect on all three types of cells via activation of two cell death mechanisms (apoptosis and necrosis). Complex 2 exhibited better potency and efficacy than the canonical cytotoxic drug cisplatin. Moreover, during shorter incubations, complex 2 demonstrated a significant SOD mimetic activity, and it was more effective and more potent than the well-known SOD mimetic TEMPOL. In addition, complex 2 was able to interact with DNA and, cleave DNA in the presence of sodium ascorbate. This study shows the potential of using polynuclear redox active compounds for developing novel anticancer drugs through SOD-mimetic redox pathways.
Assuntos
Mimetismo Biológico , Complexos de Coordenação/farmacologia , Cobre/química , Superóxido Dismutase/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Estabilidade de Medicamentos , Eletroforese em Gel de Ágar , Humanos , Ferro/química , Camundongos , Estrutura Molecular , Ratos , Superóxido Dismutase/química , Água/químicaRESUMO
Copper plays a key role in all living organisms by serving as a cofactor for a large variety of proteins and enzymes involved in electron transfer, oxidase and oxygenase activities, and the detoxification of oxygen radicals. Due to its toxicity, a conserved homeostasis mechanism is required. In E. coli, the CusCFBA efflux system is a copper-regulating system and is responsible for transferring Cu(I) and Ag(I) out of the periplasm domain into the extracellular domain. Two of the components of this efflux system, the CusF metallochaperone and the N-terminal domain of CusB, have been thought to play significant roles in the function of this efflux system. Resolving the metal ion transport mechanism through this efflux system is vital for understanding metal- and multidrug-resistant microorganisms. This work explores one aspect of the E. coli resistance mechanism by observing the interaction between the N-terminal domain of CusB and the CusF protein, using electron paramagnetic resonance (EPR) spectroscopy, circular dichroism (CD), and chemical cross-linking. The data summarized here show that M36 and M38 of CusB are important residues for both the Cu(I) coordination to the CusB N-terminal domain and the interaction with CusF, and K32 is essential for the interaction with CusF. In contrast, the K29 residue is less consequential for the interaction with CusF, whereas M21 is mostly important for the proper interaction with CusF.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metalochaperonas/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cobre , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/química , Proteínas de Escherichia coli/química , Lisina/análise , Lisina/metabolismo , Proteínas de Membrana Transportadoras/química , Metalochaperonas/química , Metionina/análise , Metionina/metabolismo , Modelos Moleculares , Mapeamento de Interação de Proteínas , Estrutura Terciária de ProteínaRESUMO
We tested the antiproliferative activity and mechanism of the action of several novel aminoacridine derivatives. Six different cancer cell lines were used to evaluate the potential cytotoxic effect of eleven aminoacridine-based molecules. A standard MTT assay was used for cell bioavailability analysis. Additionally, the potential cytotoxic effect of the tested compounds on non-cancer cells was investigated in rat skeletal muscle myotubes (L6) and in bovine aortic smooth muscle cells. In order to investigate whether the DNA binding activity of tested compounds correlated with their cytotoxic effect, circular dichroism (CD) measurement and DNA T4 ligase assay were performed. Finally, the potential mutagenic activity of the lead compound 5 was investigated. The cytotoxic effect of compound 5 in cancer cells was obtained in lower concentrations than the well-known: 9- aminoacridine based drug, amsacrine. The lead compound binds to DNA, but in a different mode than the parent molecules. Additionally, compound 5 was not cytotoxic in the effective range of concentrations in non-cancer cells. In identical concentrations, the parent compound (9-aminoacridine) and amsacrine were extremely toxic for both types of these normal cells. Finally, based on CD measurement and T4 ligase assay, it was confirmed that 5 binds to DNA but in different from the parent compounds manner. Important to mention, that compound 5 might have increased mutagenic activity which must be verified in vivo. Based on these in vitro results, we conclude that 5 is a more potent and more selective antiprolifirative compound than amsacrine. Compound 5 was also more effective in HepG2 and P-12 cells. Thus, 5 is suitable for future in vivo biological evaluation and its structure might be used as a basis for developing novel anticancer drugs.
Assuntos
Aminoacridinas/síntese química , Antineoplásicos/síntese química , Substâncias Intercalantes/farmacologia , Aminoacridinas/farmacologia , Amsacrina/química , Amsacrina/toxicidade , Animais , Antineoplásicos/farmacologia , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/antagonistas & inibidores , DNA/química , DNA Ligase Dependente de ATP , DNA Ligases/química , Humanos , Substâncias Intercalantes/química , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Especificidade de Órgãos , Ratos , Relação Estrutura-AtividadeRESUMO
Both the essentiality and the toxicity of copper in human, yeast, and bacteria cells require precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. This work explores one aspect in the copper cycle, by probing the interaction between the human copper chaperone Atox1 and the c-terminal domain of the copper transporter, CTR1, using electron paramagnetic resonance (EPR) spectroscopy and circular dichroism (CD). The data collected here shows that the Atox1 keeps its dimer nature also in the presence of the CTR1 c-terminal domain; however, two geometrical states are assumed by the Atox1. One is similar to the geometrical state reported by the crystal structure, while the latter has not yet been constructed. In the presence of the CTR1 c-terminal domain, both states are assumed; however, the structure of Atox1 is more restricted in the presence of the CTR1 c-terminal domain. This study also shows that the last three amino acids of the CTR1 c-terminal domain, HCH, are important for maintaining the crystal structure of the Atox1, allowing less structural flexibility and improved thermal stability of Atox1.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metalochaperonas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Cobre/metabolismo , Proteínas de Transporte de Cobre , Transportador de Cobre 1 , Humanos , Metalochaperonas/química , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.
Assuntos
Flexiviridae/fisiologia , Proteínas do Movimento Viral em Plantas/análise , Plasmodesmos/química , Replicação Viral , Flexiviridae/genética , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células Vegetais/química , Células Vegetais/ultraestrutura , Doenças das Plantas/virologia , Folhas de Planta/virologia , Proteínas do Movimento Viral em Plantas/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Nicotiana/virologia , Vitis/virologiaRESUMO
Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Gênero Iris/química , Triterpenos/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Acroleína/análogos & derivados , Acroleína/isolamento & purificação , Acroleína/farmacologia , Amidas/farmacologia , Animais , Adesão Celular , Tamanho Celular , Cicloexanóis/isolamento & purificação , Cicloexanóis/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Piridinas/farmacologia , Ratos , Rizoma/química , Transfecção , Triterpenos/química , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Grapevine virus A (GVA) is closely associated with the economically important rugose-wood disease of grapevine. In an attempt to develop GVA resistance, we made a GFP-tagged GVA-minireplicon and utilized it as a tool to consistently activate RNA silencing. Launching the GVA-minireplicon by agroinfiltration delivery resulted in a strong RNA silencing response. In light of this finding, we produced transgenic Nicotiana benthamiana plants expressing the GVA-minireplicon, which displayed phenotypes that could be attributed to reproducibly and consistently activate post-transcriptional gene silencing (PTGS). These included: (i) low accumulation of the minireplicon-derived transgene; (ii) low GFP expression that was increased upon agroinfiltration delivery of viral suppressors of silencing; and (iii) resistance against GVA infection, which was found in 60%, and in 90-95%, of T1 and T2 progenies, respectively. A grafting assay revealed that non-silenced scions exhibited GVA resistance when they were grafted onto silenced rootstocks, suggesting transmission of RNA silencing from silenced rootstocks to non-silenced scions. Despite being extremely resistant to GVA infection, the transgenic plants were susceptible to the closely related vitivirus, GVB. Furthermore, infection of the silenced plants with GVB or Potato virus Y (PVY) resulted in suppression of the GVA-specific defense. From these data we conclude that GVA-minireplicon-mediated RNA silencing provides an important and efficient approach for consistent activation of PTGS that can be used for controlling grapevine viruses. However, application of this strategy for virus resistance necessitates consideration of possible infection by other viruses.
Assuntos
Nicotiana/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/genética , Interferência de RNA , Vírus de Plantas/fisiologia , Plantas Geneticamente Modificadas/virologia , Potyvirus/fisiologia , Nicotiana/virologiaRESUMO
Virus-induced gene silencing (VIGS) is an attractive approach for studying gene function. Although the number of virus vectors available for use in VIGS experiments has increased in recent years, most of these vectors are applied in annual or herbaceous plants. The aim of this work was to develop a VIGS vector based on the Grapevine virus A (GVA), which is a member of the genus Vitivirus, family Flexiviridae. The GVA vector was used to silence the endogenous phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. In addition, an Agrobacterium-mediated method for inoculating micropropagated Vitis vinifera cv. Prime plantlets via their roots was developed. Using this method, it was possible to silence the endogenous PDS gene in V. vinifera plantlets. The GVA-derived VIGS vector may constitute an important tool for improving functional genomics in V. vinifera.