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1.
Int J Mol Sci ; 20(22)2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31752214

RESUMO

'Candidatus Liberibacter asiaticus' (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H2O2 accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants.


Assuntos
Proteínas de Bactérias/genética , Rhizobiaceae/metabolismo , Tabaco/citologia , Apoptose , Proteínas de Bactérias/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Mutação , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , Sinais Direcionadores de Proteínas , Tabaco/genética , Tabaco/metabolismo , Transfecção
2.
Int J Mol Sci ; 20(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540359

RESUMO

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.


Assuntos
Proteínas de Bactérias/genética , Malus/microbiologia , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Tabaco/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Sobrevivência Celular , Expressão Gênica , Interações Hospedeiro-Patógeno , Malus/citologia , Phytoplasma/química , Phytoplasma/genética , Phytoplasma/patogenicidade , Protoplastos/citologia , Protoplastos/microbiologia , Alinhamento de Sequência , Tabaco/citologia , Fatores de Virulência/análise , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
IET Nanobiotechnol ; 13(6): 609-616, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31432794

RESUMO

Here, a rapid and easy transformation by electroporation technique for gene transfer in plants using cell penetrating amino nanocomplex (nanoplex) has been demonstrated in Nicotiana. Nanoplex was prepared using cell penetrating amino acids (CPAs) such as poly-L-lysine (PLL) and Argenine (Arg), in combination with the gold nanoparticles (AuNPs). PLLs-modified nanoplex with zeta potential of 34.2 ± 1.22 mV charge showed 63.3% efficiency for gene transformation in plant cells as compared to 60% when modified with Arg and the zeta potential was found to be 30.0 ± 0.83 mV; whereas, the transformation efficiency without nanoplex was found to be 6.6%. The findings indicate that the zeta potential of positively charged nanocomplex (AuNPs/CPAs/DNA/CPAs) increases the transformation efficiency because of their ability to protect the DNA from electroporation wave and endogenous enzyme damage. Transformation was confirmed by GUS assay and amplification of npt gene. This technique may open up new possibilities of gene transfer in plants, which will enable to produce large number of transgenic plants.


Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Ouro/química , Nanopartículas Metálicas/química , Plantas/genética , Transformação Genética/fisiologia , Agrobacterium tumefaciens , Células Cultivadas , DNA de Plantas/genética , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Tabaco/citologia , Tabaco/genética
4.
Molecules ; 24(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426346

RESUMO

Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. They were transformed by cultured Nicotiana tabacum cells to 7-ß-glucoside and 7-ß-gentiobioside of daidzein, and 3'- and 7-ß-glucosides, 3',7-ß-diglucoside, and 7-ß-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with ß-glucosidase to give 4'- and 7-ß-galactosides of daidzein, which were new compounds, and α-tocopherol 6-ß-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.


Assuntos
Antialérgicos/síntese química , Cosméticos/síntese química , Glicosídeos/síntese química , Hesperidina/síntese química , Isoflavonas/síntese química , alfa-Tocoferol/síntese química , Animais , Antialérgicos/metabolismo , Biocatálise , Técnicas de Cultura de Células , Cosméticos/metabolismo , Alimento Funcional/análise , Glicosídeos/metabolismo , Glicosilação , Hesperidina/metabolismo , Humanos , Isoflavonas/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Células Vegetais/metabolismo , Cultura Primária de Células , Ratos , Ratos Wistar , Solubilidade , Tabaco/citologia , Tabaco/metabolismo , alfa-Tocoferol/metabolismo
5.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370181

RESUMO

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.


Assuntos
Fragmentos de Peptídeos/química , Polissacarídeos/química , Proteínas do Envelope Viral/química , Sulfato de Amônio/química , Animais , Células CHO , Sequência de Carboidratos , Precipitação Química , Cromatografia em Gel/métodos , Cricetulus , Galactose/química , Expressão Gênica , Glicosilação , Humanos , Ácido N-Acetilneuramínico/química , Fragmentos de Peptídeos/isolamento & purificação , Células Vegetais/metabolismo , Polissacarídeos/isolamento & purificação , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
6.
Plant Physiol Biochem ; 142: 34-42, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255907

RESUMO

The 24-kDa protein (p24) encoded by Grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor (RSS), but its effect on active viral infection is unclear. Using a Potato virus X (PVX)-based expression system, we demonstrated that p24 elicits lethal systemic necrosis in Nicotiana benthamiana, sharing typical characteristics of the hypersensitive response (HR), and that NbRAR1 (a cytoplasmic Zn2+-binding protein) is involved in the PVX-p24-mediated systemic necrosis. Moreover, expression of p24 from Barley stripe mosaic virus (BSMV) vector triggered local necrosis in infiltrated patches of N. benthamiana, likely inhibiting viral systemic spread. By deletion analysis, we demonstrated that amino acids (aa) 1 to 180, which are located in the region (aa 1-188) previously shown to be necessary for p24's RSS activity, is sufficient for p24 to elicit systemic necrosis in the context of PVX infection. Using substitution mutants, we revealed that silencing-suppression-defective mutants R2A and W54A induce only a mild necrotic response; two mutants without self-interaction ability previously shown to lose or retain weak suppression function also displayed decreased pathogenicity: W149A without RSS activity elicited a mild necrotic response, whereas V162H/L169H/L170H which retains weak RSS activity was able to induce systemic necrosis, but with a 1- to 2-day delay. Taken together, p24 plays an important role in GLRaV-2 pathogenesis, triggering HR-like necrosis in N. benthamiana plants when expressed from PVX or BSMV vector; both the silencing suppression and self-interaction are crucial for p24's pathogenicity activity, and the region of p24 for determining systemic necrosis is mapped to aa 1-180.


Assuntos
Closterovirus/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Potexvirus/genética , Tabaco/virologia , Proteínas Virais/genética , Morte Celular , Closterovirus/patogenicidade , Regulação Viral da Expressão Gênica , Inativação Gênica , Interações Hospedeiro-Patógeno/genética , Mutação , Folhas de Planta/genética , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vírus de Plantas/patogenicidade , Potexvirus/patogenicidade , Tabaco/citologia , Proteínas Virais/metabolismo
7.
Int J Biol Macromol ; 137: 1286-1297, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31252017

RESUMO

A neutral polysaccharide separated from Dendrobium nobile Lindl was designated as DNPE6(4). It was structurally characterized using a combination of spectral and chemical analysis. Its average molecule weight was 99.2 kDa. The monosaccharide composition was Araf, Glcp, Galp, and Manp in a molar ratio of 2.5:0.9:0.3:0.8. Their linkage types were →1)-L-Araf-(3→, →1)-D-Glcp-(4→, →1)-D-Galp-(3→, →1)-D-Galp-(6→, →1)-D-Manp-(3, 6→, and T-D-Manp. The polysaccharide was found to have anti-TMV and anti-CMV activities for the first time in vivo. Notably, DNPE6(4) exhibited excellent protective activity against TMV. Furthermore, several proteins related to calcium signaling pathway and pathogen related proteins were up-regulated, and we also found expression levels of EDS1, ICS1, and PR1 involved in SA pathway up-regulated after DNPE6(4) treatment. In addition, some defense enzymes increased in the same condition. All these findings revealed DNPE6(4) was an elicitor to stimulate calcium signaling pathway to enhance the tobacco defense against TMV. This study therefore revealed that DNPE6(4) was a promising antiviral agent for future study.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Dendrobium/química , Polissacarídeos/farmacologia , Tabaco/efeitos dos fármacos , Tabaco/virologia , Tombusviridae/fisiologia , Antivirais/química , Antivirais/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peso Molecular , Monossacarídeos/análise , Polissacarídeos/química , Proteômica , Tabaco/citologia , Tabaco/imunologia
8.
BMC Biotechnol ; 19(1): 36, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208390

RESUMO

BACKGROUND: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. RESULTS: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. CONCLUSIONS: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Mutação , Folhas de Planta/citologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Protoplastos/citologia , Tabaco/citologia , Tabaco/genética
9.
Planta ; 250(4): 1111-1129, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31172343

RESUMO

MAIN CONCLUSION: The roles of microRNA-mediated epigenetic regulation were highlighted in the bud dormancy-activity cycle, implying that certain differentially expressed miRNAs play crucial roles in apical bud burst, such as csn-miR319c/TCP2. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by targeting mRNA transcripts for cleavage or directing translational inhibition. To investigate whether miRNAs regulate bud dormancy-activation transition in tea plant, which largely affects the yield and price of tea products and adaptability of tea trees, we constructed small RNA libraries from three different periods of bud dormancy-burst transition. Through sequencing analysis, 262 conserved and 83 novel miRNAs were identified, including 118 differentially expressed miRNAs. Quantitative RT-PCR results for randomly selected miRNAs exhibited that our comprehensive analysis is highly reliable and accurate. The content of caffeine increased continuously from the endodormancy bud to flushing bud, and differentially expressed miRNAs coupling with their targets associated with bud burst were identified. Remarkably, csn-miR319c was downregulated significantly from the quiescent bud to burst bud, while its target gene CsnTCP2 (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR 2) displayed opposite expression patterns. Co-transformation experiment in tobacco demonstrated that csn-miR319c can significantly suppress the functions of CsnTCP2. This study on miRNAs and the recognition of target genes could provide new insights into the molecular mechanism of the bud dormancy-activation transition in tea plant.


Assuntos
Camellia sinensis/genética , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , Sequência de Aminoácidos , Camellia sinensis/crescimento & desenvolvimento , Regulação para Baixo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Filogenia , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Alinhamento de Sequência , Tabaco/citologia , Tabaco/genética
10.
Methods Mol Biol ; 1992: 151-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148037

RESUMO

The microtubule cytoskeleton plays important roles in cell morphogenesis. To investigate the mechanisms of cytoskeletal organization, for example, during growth or development, in genetic studies, or in response to environmental stimuli, image analysis tools for quantitative assessment are needed. Here, we present a method for texture measure-based quantification and comparative analysis of global microtubule cytoskeleton patterns and subsequent visualization of output data. In contrast to other approaches that focus on the extraction of individual cytoskeletal fibers and analysis of their orientation relative to the growth axis, CytoskeletonAnalyzer2D quantifies cytoskeletal organization based on the analysis of local binary patterns. CytoskeletonAnalyzer2D thus is particularly well suited to study cytoskeletal organization in cells where individual fibers are difficult to extract or which lack a clearly defined growth axis, such as leaf epidermal pavement cells. The tool is available as ImageJ plugin and can be combined with publicly available software and tools, such as R and Cytoscape, to visualize similarity networks of cytoskeletal patterns.


Assuntos
Citoesqueleto/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microtúbulos/ultraestrutura , Imagem Óptica/métodos , Tabaco/citologia , Actinas/ultraestrutura , Microscopia Confocal/métodos , Software , Tabaco/ultraestrutura
11.
Methods Mol Biol ; 1992: 173-187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148038

RESUMO

FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.


Assuntos
Arabidopsis/citologia , Endocitose , Microscopia de Fluorescência/métodos , Raízes de Plantas/citologia , Tabaco/citologia , Arabidopsis/ultraestrutura , Membrana Celular/ultraestrutura , Corantes Fluorescentes/análise , Imagem Óptica/métodos , Raízes de Plantas/ultraestrutura , Tabaco/ultraestrutura
12.
Methods Mol Biol ; 1992: 189-199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148039

RESUMO

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.


Assuntos
Corantes Fluorescentes/análise , Microscopia de Fluorescência/métodos , Fosfolipídeos/análise , Células Vegetais/química , Tabaco/citologia , Corantes Fluorescentes/metabolismo , Expressão Gênica , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia Confocal/métodos , Fosfatidilinositóis/análise , Fosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Células Vegetais/metabolismo , Pólen/química , Pólen/genética , Tabaco/química , Tabaco/genética , Transformação Genética
13.
Methods Mol Biol ; 1992: 367-376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148052

RESUMO

Here we provide an overview of procedures for long-term cultivation, phenotyping, genotyping, and genetic transformation of cell cultures of tobacco cell lines BY-2 and VBI-0, and of A. thaliana, ecotype Landsberg erecta (LE) cell line. Notably, we present an improved protocol for BY-2 transformation and cloning and extend the available plant cell lines methodology toward high-throughput technologies like fluorescent-based cell sorting and transcriptomics.


Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Tabaco/citologia , Tabaco/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular , Clonagem Molecular/métodos , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Técnicas de Genotipagem/métodos , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Transcriptoma , Transformação Genética
14.
Methods Mol Biol ; 1991: 33-42, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041760

RESUMO

In plants, RNA silencing is an important mechanism for gene regulation and defense that is targeted by proteins of viral pathogens effecting silencing suppression. In this chapter we describe a new assay to probe silencing suppressor activity using Agrobacterium infiltration of Nicotiana benthamiana and confocal microscopy. The key element in this assay involves the use of a reporter construct that is transiently expressed at a much lower level than free GFP, and this increases the sensitivity of detection of weak silencing suppressors such as the P6 protein of Cauliflower mosaic virus. Although initially developed for virus silencing suppressors, this technique could also prove valuable to characterize the potential for weak silencing suppressors in the effector repertoires of fungi, bacteria, nematodes, and oomycetes.


Assuntos
Agrobacterium/fisiologia , Proteínas de Ligação a DNA/metabolismo , Microscopia Confocal/métodos , Doenças das Plantas/virologia , Interferência de RNA , Tabaco/citologia , Tabaco/genética , Proteínas Virais/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/metabolismo , Supressão Genética , Tabaco/virologia , Proteínas Virais/genética
15.
Plant Mol Biol ; 100(3): 215-230, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053988

RESUMO

KEY MESSAGE: Two homologs PsnSuSy1 and PsnSuSy2 from poplar played largely similar but little distinct roles in modulating sink strength, accelerating vegetative growth and modifying secondary growth of plant. Co-overexpression of them together resulted in small but perceptible additive effects. Sucrose synthase (SuSy) acts as a crucial determinant of sink strength by controlling the conversion of sucrose into UDP-glucose, which is not only the sole precursor for cellulose biosynthesis but also an extracellular signaling molecule for plants growth. Therefore, modification of SuSy activity in plants is of utmost importance. We have isolated two SuSy genes from poplar, PsnSuSy1 and PsnSuSy2, which were preferentially expressed in secondary xylem/phloem. To investigate their functions, T2 tobacco transgenic lines of PsnSuSy1 and PsnSuSy2 were generated and then crossed to generate PsnSuSy1/PsnSuSy2 dual overexpression transgenic lines. SuSy activities in all lines were significantly increased though PsnSuSy1/PsnSuSy2 lines only exhibited slightly higher SuSy activities than either PsnSuSy1 or PsnSuSy2 lines. The significantly increased fructose and glucose, engendered by augmented SuSy activities, caused the alternations of many physiological, biochemical measures and phenotypic traits that include accelerated vegetative growth, thickened secondary cell wall, and increased stem breaking force, accompanied with altered expression levels of related pathway genes. The correlation relationships between SuSy activities and many of these traits were statistically significant. However, differences of almost all traits among three types of transgenic lines were insignificant. These findings clearly demonstrated that PsnSuSy1 and PsnSuSy2 had similar but little distinct functions and insubstantial additive effects on modulating sink strength and affecting allocation of carbon elements among secondary cell wall components.


Assuntos
Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucosiltransferases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Parede Celular/ultraestrutura , Celulose/biossíntese , Clorofila/análise , Clonagem Molecular , Perfilação da Expressão Gênica , Glucosiltransferases/metabolismo , Lignina/metabolismo , Floema/metabolismo , Fotossíntese , Polissacarídeos/metabolismo , Populus/genética , Análise de Sequência , Sacarose/metabolismo , Tabaco/citologia , Tabaco/crescimento & desenvolvimento , Xilema/metabolismo
16.
Biochemistry ; 58(16): 2188-2197, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30942568

RESUMO

In this study, our fundamental research interest was to understand how negatively charged porphyrins could interact with a plant cell wall and further act inside cells. Thus, three anionic porphyrins differing in their anionic external groups (carboxylates, sulfonates, and phosphonates) were tested. First, the tobacco cell wall was isolated to monitor in vitro its interactions with the three different anionic porphyrins. Unexpectedly, these negatively charged molecules were able to bind to the negatively charged cell wall probably by weak bonds such as hydrogen bonds and/or electrostatic interactions when the tetrapyrrolic core was protonated. Moreover, we showed that at the pH of spent culture medium (4.5), the neutrality of the carboxylated porphyrin (TPPC) facilitated its cell wall crossing while the diffusion of the two other sulfonated (TPPS) or phosphonated (TPPP) porphyrins that remained anionic was delayed. Once inside Tobacco Bright Yellow-2 (TBY-2) cells, TPPC induced higher levels of production of both H2O2 and malondialdehyde compared to TPPS after illumination. That result correlated well with strong cell death induction by photoactivated TPPC. Furthermore, reactive oxygen species-scavenging enzymes such as catalase, peroxidases, and superoxide dismutase were also strongly downmodulated in response to TPPC, while these enzymes were almost unchanged in response to photoactivated TPPS. To the best of our knowledge, this is the first study that took into account the whole story from interactions of porphyrins with a plant cell wall to their photodynamic activity inside the cells.


Assuntos
Ânions/química , Parede Celular/metabolismo , Fármacos Fotossensibilizantes/química , Porfirinas/química , Ânions/metabolismo , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Células Cultivadas , Ligação de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Luz , Malondialdeído/metabolismo , Estrutura Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/metabolismo , Sulfonas/química , Sulfonas/metabolismo , Tabaco/citologia , Tabaco/efeitos dos fármacos , Tabaco/metabolismo
17.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
18.
Planta ; 250(1): 79-94, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919065

RESUMO

MAIN CONCLUSION: Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Sesquiterpenos/metabolismo , Tabaco/metabolismo , Triglicerídeos/metabolismo , Animais , Vias Biossintéticas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/química , Proteínas de Membrana/genética , Camundongos , Especificidade de Órgãos , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteômica , Sesquiterpenos/análise , Terpenos/metabolismo , Tabaco/citologia , Tabaco/genética , Triglicerídeos/análise
19.
Plant Physiol ; 180(1): 654-681, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30862726

RESUMO

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.


Assuntos
Folhas de Planta/citologia , Folhas de Planta/fisiologia , Biologia de Sistemas/métodos , Tabaco/citologia , Aminoácidos/metabolismo , Metabolismo dos Carboidratos , Núcleo Celular/genética , Cloroplastos , Genomas de Plastídeos , Luz , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Fotossíntese , Plastídeos/genética , Tabaco/fisiologia , Transcriptoma , Triglicerídeos/metabolismo
20.
J Biotechnol ; 295: 41-48, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30826446

RESUMO

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.


Assuntos
Vacinas contra Ebola , Ebolavirus/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Proteínas do Envelope Viral , Células Cultivadas , Vacinas contra Ebola/química , Vacinas contra Ebola/genética , Vacinas contra Ebola/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vegetais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
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