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1.
Mol Plant Microbe Interact ; 33(2): 166-172, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855496

RESUMO

Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.


Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Células Vegetais , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Células Vegetais/microbiologia , Plasmídeos/genética , Domínios Proteicos
2.
J Agric Food Chem ; 67(49): 13518-13525, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31757125

RESUMO

Coordinating unsaturated metal sites (CUS) on the surface of metal-organic frameworks (MOFs) could be used to adsorb His-tagged proteins. The specific adsorption between CUS and His-tagged proteins could reduce preparation steps, shorten preparation time, and could also avoid the binding between the metal ion of metalloenzyme active center and the chelating agent to ensure the enzyme activity. In this study, MIL-88A was synthesized by hydrothermal method and used to purify and immobilize His-tagged organophosphohydrolase (OpdA) in one step for organophosphate bioremediation. Under optimized conditions, OpdA@MIL-88A had a maximal activity of 1554 U/gprotein, which was nearly 5 times higher than free OpdA. Compared with free OpdA, OpdA@MIL-88A exhibited improved organic solvent tolerance, SDS tolerance, thermal stability, and storage stability. OpdA@MIL-88A was used to degrade organophosphorus pesticides on grapes and cucumbers. After reuse 6 times, OpdA@MIL-88A retained more than 66% and 61% of the initial activity, respectively. Therefore, this proposed strategy provided a facile and effective method for degradation of organophosphorus pesticides.


Assuntos
Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Estruturas Metalorgânicas/química , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Monoéster Fosfórico Hidrolases/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Meios de Cultura/química , Meios de Cultura/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Compostos Organofosforados/química , Praguicidas/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
4.
Microbiol Res ; 226: 10-18, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284939

RESUMO

Microbial oxidation of antimonite [Sb(III)] to antimonate [Sb(V)] is a detoxification process which contributes to Sb(III) resistance. Antimonite oxidase AnoA is essential for Sb(III) oxidation, however, the regulation mechanism is still unknown. Recently, we found that the expressions of phosphate transporters were induced by Sb(III) using proteomics analysis in Agrobacterium tumefaciens GW4, thus, we predicted that the phosphate regulator PhoB may regulate bacterial Sb(III) oxidation and resistance. In this study, comprehensive analyses were performed and the results showed that (1) Genomic analysis revealed two phoB (named as phoB1 and phoB2) and one phoR gene in strain GW4; (2) Reporter gene assay showed that both phoB1 and phoB2 were induced in low phosphate condition (50 µM), but only phoB2 was induced by Sb(III); (3) Genes knock-out/complementation, Sb(III) oxidation and Sb(III) resistance tests showed that deletion of phoB2 significantly inhibited the expression of anoA and decreased bacterial Sb(III) oxidation efficiency and Sb(III) resistant. In contrast, deletion of phoB1 did not obviously affect anoA's expression level and Sb(III) oxidation/resistance; (4) A putative Pho motif was predicted in several A. tumefaciens strains and electrophoretic mobility shift assay (EMSA) showed that PhoB2 could bind with the promoter sequence of anoA; (5) Site-directed mutagenesis and short fragment EMSA revealed the exact DNA binding sequence for the protein-DNA interaction. These results showed that PhoB2 positively regulates Sb(III) oxidation and PhoB2 is also associated with Sb(III) resistance. Such regulation mechanism may provide a great contribution for bacterial survival in the environment with Sb and for bioremediation application.


Assuntos
Agrobacterium tumefaciens/metabolismo , Antimônio/metabolismo , Proteínas de Bactérias/metabolismo , Fosfatos/metabolismo , Agrobacterium tumefaciens/genética , Arsenitos/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas de Transporte de Fosfato/metabolismo , Proteômica
5.
Microbiol Res ; 226: 55-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284945

RESUMO

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.


Assuntos
Agrobacterium tumefaciens/genética , Ascomicetos/genética , Transformação Genética , Ascomicetos/citologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Southern Blotting , Carbenicilina/farmacologia , Técnicas de Cocultura , DNA Bacteriano , Regulação Fúngica da Expressão Gênica , Gentamicinas/farmacologia , Proteínas de Fluorescência Verde/genética , Higromicina B/farmacologia , Canamicina/farmacologia , Reação em Cadeia da Polimerase , Análise de Sequência , Virulência/genética
6.
World J Microbiol Biotechnol ; 35(8): 115, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332542

RESUMO

Antibiotic and arsenic (As) contaminations are worldwide public health problems. Previously, the bacterial ABC-type efflux protein MacAB reportedly conferred resistance to macrolide-type antibiotics but not to other metal(loid)s. In this study, the roles of MacAB for the co-resistance of different antibiotics and several metal(loid)s were analyzed in Agrobacterium tumefaciens 5A, a strain resistant to arsenite [As(III)] and several types of antibiotics. The macA and macB genes were cotranscribed, and macB was deleted in A. tumefaciens 5A and heterologously expressed in Escherichia coli AW3110 and E. coli S17-1. Compared to the wild-type strain 5A, the macB deletion strain reduced bacterial resistance levels to several macrolide-type and penicillin-type antibiotics but not to cephalosporin-type antibiotics. In addition, the macB deletion strain showed lower resistance to As(III) but not to arsenate [As(V)], antimonite [Sb(III)] and cadmium chloride [Cd(II)]. The mutant strain 5A-ΔmacB cells accumulated more As(III) than the cells of the wild-type. Furthermore, heterologous expression of MacAB in E. coli S17-1 showed that MacAB was essential for resistance to macrolide, several penicillin-type antibiotics and As(III) but not to As(V). Heterologous expression of MacAB in E. coli AW3110 reduced the cellular accumulation of As(III) but not of As(V), indicating that MacAB is responsible for the efflux of As(III). These results demonstrated that, in addition to macrolide-type antibiotics, MacAB also conferred resistance to penicillin-type antibiotics and As(III) by extruding them out of cells. This finding contributes to a better understanding of the bacterial resistance mechanisms of antibiotics and metal(loid)s.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Macrolídeos/farmacologia , Agrobacterium tumefaciens/metabolismo , Arsenitos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinas/farmacologia , DNA Bacteriano/genética , Eritromicina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Penicilinas/farmacologia
7.
Microbiology ; 165(10): 1117-1134, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31339484

RESUMO

The Agrobacterium tumefaciens ActSR two-component regulatory system is a member of a homologous group of global redox-responsive regulatory systems that adjust the expression of energy-consuming and energy-supplying metabolic pathways in order to maintain cellular redox balance. In this study, the transcriptional organization of the hrpB-actSR locus was determined and the effect of actSR system inactivation on stress resistance was investigated. It was found that hrpB is transcribed as a monocistronic mRNA and actS is transcribed along with actR as a bicistronic mRNA, while actR is also transcribed as a monocistronic message. Each message is initiated from a separate promoter. Inactivation of actR resulted in decreased resistance to membrane stress (sodium dodecyl sulfate), acid stress (pH 5.5), iron starvation (bipyridyl) and iron excess (FeCl3), and antibiotic stress (tetracycline and ciprofloxacin). Resistance to oxidative stress in the form of organic peroxide (cumene hydroperoxide) increased, while resistance to inorganic peroxide (H2O2) decreased. An actR insertion mutant displayed reduced catalase activity, even though transcription of katA and catE remained unchanged. Complementation of the actR inactivation mutant with plasmid-encoded actR or overexpression of hemH, encoding ferrochelatase, restored wild-type catalase activity and H2O2 resistance levels. Gel mobility shift and hemH promoter-lacZ fusion results indicated that ActR is a positive regulator of hemH that binds directly to the hemH promoter region. Thus, inactivation of the A. tumefaciens ActSR system affects resistance to multiple stresses, including reduced resistance to H2O2 resulting from a reduction in catalase activity due to reduced expression of hemH.


Assuntos
Adaptação Fisiológica/fisiologia , Agrobacterium tumefaciens/fisiologia , Ferroquelatase/genética , Peróxido de Hidrogênio/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catalase/metabolismo , Regulação Bacteriana da Expressão Gênica , Oxirredução , Regiões Promotoras Genéticas , Proteínas Quinases/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Transcrição Genética , Virulência
8.
Res Microbiol ; 170(6-7): 263-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31279088

RESUMO

In bacteria, phosphate (Pi) stress response is governed by the two-component regulatory system, sensor kinase PhoR and its cognate response regulatory protein PhoB. The arsenite [As(III)]-oxidizing bacterium Agrobacterium tumefaciens GW4 contains two phoB genes, phoB1 and phoB2. phoB1 is adjacent to As(III)-oxidizing genes, however, the functions of PhoB1 and PhoB2 remain unclear. Here, phoB1 and phoB2 were each deleted in-frame, and proteomics, qRT-PCR and protein-DNA interaction were performed. We found that (1) phoB1 and phoB2 were both upregulated under low Pi conditions and phoB1 was induced by As(III), but phoB2 was not; (2) deletion of phoB1 reduced As(III)-oxidizing efficiency and protein-DNA interaction analysis showed PhoB1 could interact with aioXSR promoter to regulate As(III) oxidation; (3) deletions of phoB1 or phoB2 both reduced exopolysaccharides (EPS) synthesis; and (4) PhoB1 influenced Pi uptake, As(III) oxidation, EPS synthesis, TCA cycle, energy production and stress response with As(III), and PhoB2 was associated with Pi uptake and EPS synthesis in low Pi conditions. These results showed PhoB1 and PhoB2 were both involved in Pi acquisition, PhoB1 was more important with As(III) and PhoB2 played a major role without As(III). Strain GW4 uses these two regulators to survive under low Pi and arsenic-rich environments.


Assuntos
Agrobacterium tumefaciens/genética , Arsenitos/metabolismo , Proteínas de Bactérias/genética , Fosfatos/metabolismo , Polissacarídeos Bacterianos/metabolismo , Agrobacterium tumefaciens/metabolismo , Ciclo do Ácido Cítrico/genética , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação Bacteriana da Expressão Gênica/genética , Oxirredução , Polissacarídeos Bacterianos/biossíntese
9.
Biotechnol J ; 14(10): e1900113, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31218827

RESUMO

Large-scale automated transient protein expression in plants requires the synchronization of cultivation and bacterial fermentation, especially if more than one bacterial strain. Therefore, a ready-to-use approach that decouples bacterial fermentation and infiltration is developed. It is found that bacterial cultures can easily be reconstituted in infiltration medium at a user-defined time, optical density, and quantity. This allows the process flow to be staggered, avoiding bottlenecks in process capacity and labor. Using the red fluorescent protein, DsRed, as a model product, the ready-to-use preparations achieved the same yields in infiltrated plant biomass as Agrobacterium tumefaciens derived from regular fermentations. It is possible to store the ready-to-use stocks at -20 °C and -80 °C for more than two months without loss of activity. Using a consolidated cost model for the current fermentation process, it is found that the ready-to-use strategy can reduce operational costs by 20-95% and investment costs by up to 75%, which would otherwise offset the economic advantages of plants over mammalian expression systems during upstream production. Furthermore, the staggered cultivation of plants and bacteria reduces the likelihood of batch failure and thus increases the robustness and flexibility of transient expression for the production of recombinant proteins in plants.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas Recombinantes/metabolismo , Tabaco/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Criopreservação , Fermentação , Regulação da Expressão Gênica de Plantas , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Viabilidade Microbiana , Proteínas Recombinantes/genética , Tabaco/genética , Tabaco/microbiologia
10.
Genes (Basel) ; 10(6)2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248134

RESUMO

The establishment of genetic transformation method is crucial for the functional genomics research in filamentous fungi. Although the transformation method has been developed in several types of fungi, a highly efficient and convenient transformation system is desperately needed in Lentinula edodes. Present work established the Agrobacterium-mediated transformation (ATMT) of basidiomycete L. edodes in both monokaryon and dikaryon mycelia by using constructed binary plasmid pCAMBIA-1300-GFP. Then, the transformation efficiency of ATMT was evaluated by using different mediums for recipient incubation and different varieties of L. edodes. The results showed that in dikaryon strain W1, the positive hygromycin-resistant transformants was observed in all medium with the positive frequency of selected transformants that ranged from 0 to 30%. While in the monokaryon strain W1-26, only the millet medium group obtained positive transformants with a positive frequency of 75.48%. Moreover, three dikaryotic wild strains (YS55, YS3334, and YS3357) and two dikaryotic cultivated strains (W1 and S606) showed the highest transformation efficiency, with 32.96% of the germination frequency, and 85.12% of positive frequency for hygromycin-resistant transformants. This work demonstrated that Agrobacterium-mediated transformation was successfully performed in L. edodes, and the genotype of recipients as well as the medium for mycelial incubation were suggested to play key roles in determining the transformation efficiency. These findings may provide new avenues for the genetic modification of edible mushroom and may extend the cognition of DNA-mediated transformation in filamentous fungi.


Assuntos
Agaricales/genética , Agrobacterium tumefaciens/genética , Cogumelos Shiitake/genética , Transformação Genética , Basidiomycota/genética , Patrimônio Genético , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Micélio/genética , Plasmídeos/genética
11.
GM Crops Food ; 10(2): 115-138, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31187675

RESUMO

An efficient genetic transformation system is a prerequisite for studying gene functions, molecular breeding program, and introducing new traits. Agrobacterium tumefaciens-mediated genetic transformation is a widely preferred and accepted method for many plants, including pigeon pea. However, the efficiency of transformation of pigeon pea using the existing protocols is low and time-consuming. In the present study, we developed a rapid and highly efficient transformation system of pigeon pea, using embryonic axis-attached cotyledons as explants. We systematically investigated the influence of varying optical densities of Agrobacterium suspension, duration of incubation, and co-cultivation on the transformation efficiency. In our system, a transformation efficiency of approximately 83% was achieved using Agrobacterium cells at an optical density (OD600) of 0.25, infection time of 15 min, and co-culturing with explants for 72 h in the light with 100µM acetosyringone. The entire procedure, starting from seed to establishment of transformed plants in soil, was achieved in 35-40 days. This is a rapid and highly efficient protocol for Agrobacterium-mediated transformation of pigeon pea, which could potentially be a useful reference, not only for the genetic improvement of pigeon pea but also for other recalcitrant leguminous plants.


Assuntos
Agrobacterium tumefaciens/genética , Cajanus/genética , Transformação Genética/genética , Cotilédone/genética , Cotilédone/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
12.
Appl Microbiol Biotechnol ; 103(14): 5763-5780, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31127355

RESUMO

The expression of the Agrobacterium tumefaciens emrAB operon, which encodes a membrane fusion protein and an inner membrane protein, is inducible by various flavonoids, including apigenin, genistein, luteolin, naringenin, and quercetin. Among these flavonoids, quercetin is the best inducer, followed by genistein. The emrR gene is divergently transcribed from the emrAB operon. The EmrR protein, which belongs to the TetR transcriptional regulator family, negatively regulates the expression of emrAB and of itself. Electrophoretic mobility shift assays and DNase I footprinting showed that EmrR binds directly at two EmrR-binding sites in the emrR-emrAB intergenic region and that quercetin inhibits the DNA-binding activity of EmrR. Promoter-lacZ fusion analyses and 5' rapid amplification of cDNA ends were performed to map the emrR and emrAB promoters. Compared with the wild-type strain, the emrA mutant strain exhibited similar levels of resistance to the tested antibiotics. In contrast, disruption of emrR conferred protection against nalidixic acid and novobiocin, but it rendered A. tumefaciens sensitive to tetracycline and erythromycin. The emrR mutation also destabilized the outer membrane of A. tumefaciens, resulting in increased sensitivity to SDS and low pH. These findings demonstrate that proper regulation of emrR-emrAB is required for free-living A. tumefaciens to survive in deleterious environments in which toxic compounds are present. Nonetheless, A. tumefaciens strains that lack emrR or emrA still have the ability to cause tumors when infecting Nicotiana benthamiana plants.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/metabolismo , Flavonoides/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Agrobacterium tumefaciens/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Membrana/genética , Novobiocina/farmacologia , Óperon , Regiões Promotoras Genéticas , Tetraciclina/farmacologia , Tabaco/microbiologia
13.
Plant Physiol Biochem ; 141: 40-50, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128562

RESUMO

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.


Assuntos
Agrobacterium tumefaciens/genética , Azacitidina/farmacologia , Técnicas de Transferência de Genes , Brotos de Planta/efeitos dos fármacos , Soja/efeitos dos fármacos , Cotilédone/genética , Metilação de DNA , Fabaceae/genética , Genes de Plantas , Genótipo , Metilação , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas/genética , Protoplastos , Regeneração , Análise de Sequência de DNA , Soja/genética , Transfecção , Transformação Genética , Transgenes
14.
Int J Mol Sci ; 20(10)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137806

RESUMO

Transgenic technology is a powerful tool for gene functional characterization, and poplar is a model system for genetic transformation of perennial woody plants. However, the poplar genetic transformation system is limited to a number of model genotypes. Herein, we developed a transformation system based on efficient Agrobacterium-mediated transformation for the hybrid poplar Populus Alba × Populus glandulosa Uyeki, which is a fast-growing poplar species that is suitably grown in the northern part of China. Importantly, we optimized many independent factors and showed that the transformation efficiency was improved significantly using juvenile leaf explants. Explants were infected by an Agrobacterium suspension with the OD600 = 0.6 for 15 min and then co-cultured in dark conditions for 3 days. Using the improved transformation system, we obtained the transgenic poplar with overexpression of ß-glucuronidase (GUS) via direct organogenesis without callus induction. Furthermore, we analyzed the GUS gene in the transgenic poplars using PCR, qRT-PCR, and GUS staining. These analyses revealed that the GUS gene was efficiently transformed, and it exhibited various expression levels. Taken together, these results represent a simple, fast, and efficient transformation system of hybrid poplar plants. Our findings may facilitate future studies of gene functions in perennial woody plants and tree breeding via transgenic technology assisted design.


Assuntos
Agrobacterium tumefaciens/genética , Plantas Geneticamente Modificadas/genética , Populus/genética , Transformação Genética , Glucuronidase/genética , Glucuronidase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
15.
Mol Biotechnol ; 61(7): 528-540, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31065900

RESUMO

Tall fescue plants are widely exposed to white grubs. Regarding the rate of damage caused by the white grubs to tall fescue and difficulty of its ecological and economical control, production of resistant cultivars is a priority. In this experiment, for the first time, we report production of transgenic lines resistant to white grub using CR8DB gene. For this, mature seeds were placed on MS basal medium with 0-15 mg L-1 2,4-D for callogenesis and 0-1.75 mg L-1 BA for regeneration. 'Asterix' (54.11%) in 7.5 and 'Talladega' (52.53%) in 10 mg L-1 2,4-D showed maximum callogenesis. Regeneration percentage was higher in 0.5 mg L-1 BA. Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pCAMBIA 1301 with CRY8DB gene, which contains HPTII gene and uidA and various types MS media were used for transformation of calli. The highest percentage of gus enzyme activity and hygromycin resistance in calli was related to the modified medium type 11. The PCR and RT-PCR analysis was done to confirm the presence and expression of the target gene in transgenic 5 lines in 'Asterix' and 3 lines in 'Talladega'. According to bioassay, larvae mortality of 91.66% was observed in transgenic plants, whereas it was 15.52% in control plants.


Assuntos
Agrobacterium tumefaciens/genética , Toxinas Bacterianas/genética , Besouros , Festuca/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium tumefaciens/metabolismo , Animais , Toxinas Bacterianas/biossíntese , Besouros/microbiologia , Festuca/microbiologia , Proteínas de Insetos/metabolismo , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/microbiologia , Receptores de Superfície Celular/metabolismo , Técnicas de Cultura de Tecidos , Transformação Genética
16.
Plasmid ; 103: 9-16, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30928704

RESUMO

Plasmids are widely distributed in rhizobia, a group of bacteria able to establish symbiotic relationships with the roots of legume plants. Two types of conjugative transfer (CT) regulation of these elements have been described in more detail. The most prevalent is through Quorum-Sensing (QS), mediated by the interaction of the TraR regulator protein and its cognate acyl-homoserine lactone (AHL) synthesized by TraI. In this study, we analyzed rhizobial plasmids classified according to their TraR regulators into four different groups. Each group has a particular genomic architecture. In one of the groups (I-C), represented by pLPU83a from Rhizobium favelukesii LPU83, CT induction requires TraR. With manual annotation, a traI was located in the plasmid distant to the traR gene. These features make pLPU83a an interesting plasmid for studying novel mechanisms of CT regulation. We mutagenized the traI gene, and found that it does not participate in CT regulation. Furthermore, we studied whether pLPU83a is subject to QS regulation by determining CT at different growth stages (cell densities). Our results showed no positive correlation between increase in culture densities and CT induction, on the contrary a slight decrease in CT was found at higher culture densities, unlike other TraR-depending plasmids. Our results show that transfer of pLPU83a is not regulated in a QS-dependent manner, and suggest that molecules not yet identified may activate its CT. Also, accumulation of a putative inhibitor cannot be disregarded.


Assuntos
Agrobacterium tumefaciens/genética , Conjugação Genética , Plasmídeos/química , Percepção de Quorum/genética , Rhizobium/genética , Acil-Butirolactonas/metabolismo , Agrobacterium tumefaciens/metabolismo , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fabaceae/microbiologia , Anotação de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , Plasmídeos/classificação , Plasmídeos/metabolismo , Rhizobium/metabolismo , Simbiose/genética
17.
Microbiol Res ; 222: 14-24, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30928026

RESUMO

Analysis of the Agrobacterium tumefaciens C58 genome revealed a potential Zur (zinc uptake regulator) binding site (5'-GATATGTTATTACATTAC-3', the underlined letters are the center of symmetry of the inverted palindrome) located in the upstream region of atu3184, whose gene product is a member of the COG0523 subfamily of G3E GTPases. The specific interaction of the Zur protein with the 18-bp inverted repeat operator motif in the presence of zinc was demonstrated in vitro by a DNA band shift assay and a DNase I footprinting assay. A LacZ reporter fusion assay further confirmed that Zur negatively regulates atu3184 promoter activity in vivo. The expression of atu3184 was upregulated in response to zinc limitation in the wild-type strain, but the zur mutant strain exhibited high-level constitutive expression of atu3184 under all conditions, irrespective of the zinc levels. It is likely that A. tumefaciens Zur senses zinc and directly regulates the atu3184 promoter by a molecular mechanism similar to that of Escherichia coli Zur, where the operator DNA is surrounded by four Zur monomers forming two dimers bound on the opposite sides of the DNA duplex. Disruption of atu3184 did not affect cell growth under metal-limited conditions and had no effect on the total cellular zinc content. Furthermore, an A. tumefaciens strain lacking atu3184 caused a tumor disease in a host plant.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Agrobacterium tumefaciens/crescimento & desenvolvimento , Pegada de DNA , DNA Bacteriano , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares , Óperon , Regiões Promotoras Genéticas , Proteínas Recombinantes , Virulência/genética , Zinco/metabolismo
18.
Curr Protoc Plant Biol ; 4(2): e20088, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30861331

RESUMO

Brachypodium distachyon is an excellent model system for the grasses and has been adopted as a research organism by many laboratories around the world. It has all of the biological traits required for a model system, including small stature, short life cycle, small genome, simple growth requirements, and a close relationship to major crop plants (cereals). In addition, numerous resources have been developed for working with this species, including genome sequences for many lines, sequenced mutant collections, and a large, freely available germplasm collection. Fortunately, among grasses B. distachyon is one of the most easily transformed species, an absolute necessity for a model system. Agrobacterium-mediated transformation is the preferred method to transform plants because it usually results in simple insertions of target DNA. In this article, we describe a method for Agrobacterium-mediated transformation of the inbred B. distachyon lines Bd21 and Bd21-3. Embryogenic callus induced from immature embryos is co-cultivated with Agrobacterium tumefaciens strain AGL1 or Agrobacterium rhizogenes strain 18r12v. Hygromycin and paromomycin are used as selective agents, with comparable transformation efficiencies (defined as the percentage of co-cultivated callus that produce transgenic plants) of 40% to 70%. It takes 20 to 30 weeks to obtain T1 seeds starting from the initial step of dissecting out immature embryos. This protocol has been shown to be efficient and facile in several studies that resulted in the creation of over 22,000 T-DNA mutants. © 2019 by John Wiley & Sons, Inc.


Assuntos
Agrobacterium tumefaciens/genética , Brachypodium/genética , Transformação Genética , Brachypodium/crescimento & desenvolvimento , Células Cultivadas
19.
BMC Res Notes ; 12(1): 144, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30876440

RESUMO

OBJECTIVE: The treatment of plant tissue with Agrobacterium tumefaciens is often a critical first step to both stable and transient plant transformation. In both applications bacterial suspensions are oftentimes physically introduced into plant tissues using hand-driven pressure from a needleless syringe. While effective, this approach has several drawbacks that limit reproducibility. Pressure must be provided with the syringe perfectly perpendicular to the tissue surface. The researcher must also attempt to provide even and consistent pressure, both within and between experimental replicates. These factors mean that the procedures do not always translate well between research groups or biological replicates. RESULTS: We have devised a method to introduce Agrobacterium suspensions into plant leaves with greater reproducibility. Using a decommissioned dissecting microscope as an armature, a syringe body with the bacterial suspension is mounted to the nosepiece. Gentle, even pressure is applied by rotating the focus knob. The treatment force is measured using a basic kitchen scale. The development of the Standardized Pressure Agrobacterium Infiltration Device (SPAID) provides a means to deliver consistent amounts of bacterial suspensions into plant tissues with the goal of increasing reproducibility between replicates and laboratories.


Assuntos
Agrobacterium tumefaciens/genética , Arabidopsis/genética , Fragaria/genética , Folhas de Planta/genética , Tabaco/genética , Transformação Genética , Engenharia Genética/instrumentação , Engenharia Genética/métodos , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes
20.
Mol Biotechnol ; 61(5): 332-344, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30830513

RESUMO

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of 'Grassland' after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.


Assuntos
Agrobacterium tumefaciens/genética , Alquil e Aril Transferases/genética , Lolium/genética , Transfecção/métodos , Alquil e Aril Transferases/metabolismo , Temperatura Alta , Lolium/crescimento & desenvolvimento , Lolium/microbiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sonicação , Transformação Genética , Vácuo
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