RESUMO
Acremonium strictum Elicitor Subtilisin (AsES) is a fungal elicitor that activates innate immunity, conferring disease resistance in strawberry (Fragaria × ananassa Duch.), Arabidopsis and other plant species. The aim of the present work was to evaluate the involvement of the ethylene (ET) signalling pathway in AsES-mediated immune response in strawberry. Ethylene production and expression of the genes responsible for ET synthesis, perception and response were measured after AsES treatment. ROS (H2 O2 ) accumulation and immunity induced by AsES were studied after ET perception was blocked by 1-methylcyclopropene (1-MCP). Biochemical and molecular results showed that AsES induced a marked increase in local and systemic biosynthesis of ET, both in a biphasic manner. Blocking of ET perception by 1-MCP prior to AsES induction reduced production of ROS (H2 O2 ) and prevented AsES from eliciting defence against fungal pathogens having different lifestyles, such as Botrytis cinerea (necrotrophic) and Colletotrichum acutatum (hemibiotrophic). These findings contribute to elucidate the mode of action of the novel elicitor subtilase, AsES, specifically regarding the role of ET signalling in the activation of plant innate immunity, in addition to the multitude of processes regulated by ET in plants.
Assuntos
Etilenos , Fragaria , Transdução de Sinais , Subtilisina , Etilenos/metabolismo , Fragaria/efeitos dos fármacos , Fragaria/imunologia , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hypocreales/química , Transdução de Sinais/efeitos dos fármacos , Subtilisina/farmacologiaRESUMO
Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth-promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum-strawberry plant interaction. Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO3 , an inhibitor of ET biosynthesis; with 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth-promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT-PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes. Results showed that ET acts as a rapid and transient signal in the first 12 h post-treatment. A. brasilense REC3-inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up-regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling. Ethylene production and up-regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.
Assuntos
Azospirillum brasilense/metabolismo , Etilenos/metabolismo , Fragaria/microbiologia , Aminoácidos Cíclicos/farmacologia , Biomassa , Etilenos/antagonistas & inibidores , Fragaria/crescimento & desenvolvimento , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ácidos Indolacéticos/farmacologia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismo , Nitrato de Prata/farmacologiaRESUMO
The plant growth-promoting strain REC3 of Azospirillum brasilense, isolated from strawberry roots, prompts growth promotion and systemic protection against anthracnose disease in this crop. Hence, we hypothesised that A. brasilense REC3 can induce different physiological, structural and molecular responses in strawberry plants. Therefore, the aim of this work was to study these traits activated in Azospirillum-colonised strawberry plants, which have not been assessed until now. Healthy, in vitro micropropagated plants were root-inoculated with REC3 under hydroponic conditions; root and leaf tissues were sampled at different times, and oxidative burst, phenolic compound content, malondialdehyde (MDA) concentration, callose deposition, cell wall fortification and gene expression were evaluated. Azospirillum inoculation enhanced levels of soluble phenolic compounds after 12 h post-inoculation (hpi), while amounts of cell wall bound phenolics were similar in inoculated and control plants. Other early responses activated by REC3 (at 24 hpi) were a decline of lipid peroxidation and up-regulation of strawberry genes involved in defence (FaPR1), bacterial recognition (FaFLS2) and H2O2 depuration (FaCAT and FaAPXc). The last may explain the apparent absence of oxidative burst in leaves after bacterial inoculation. Also, REC3 inoculation induced delayed structural responses such as callose deposition and cell wall fortification (at 72 hpi). Results showed that A. brasilense REC3 is capable of exerting beneficial effects on strawberry plants, reinforcing their physiological and cellular characteristics, which in turns contribute to improve plant performance.
Assuntos
Azospirillum brasilense , Biomassa , Resistência à Doença , Fragaria/microbiologia , Expressão Gênica , Peroxidação de Lipídeos , Fenóis/metabolismo , Parede Celular/metabolismo , Resistência à Doença/genética , Fragaria/crescimento & desenvolvimento , Fragaria/fisiologia , Genes de Plantas , Glucanos/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Dados de Sequência Molecular , Oxirredução , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Simbiose , Regulação para CimaRESUMO
The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth-promoting bacterium Azospirillum brasilense REC3, and non-inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analysis. This allowed simultaneous semi-quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria-inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient-limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient-poor soils. SEM-EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum-inoculated and control plants had similar elemental quantities; however, in bacteria-inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).
Assuntos
Azospirillum brasilense/fisiologia , Fragaria/química , Fragaria/microbiologia , Fragaria/crescimento & desenvolvimento , Fragaria/ultraestrutura , Microscopia Eletrônica de Varredura , Folhas de Planta/química , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Espectrometria por Raios XRESUMO
Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.
Assuntos
Produtos Agrícolas/genética , Fragaria/enzimologia , Fragaria/genética , Variação Genética/genética , Imunidade Inata/genética , Doenças das Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Sequência Conservada , Produtos Agrícolas/enzimologia , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Evolução Molecular , Dados de Sequência Molecular , Filogenia , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de SequênciaRESUMO
Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria x ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry.
Assuntos
Fragaria/genética , Genes de Plantas , Variação Genética , Imunidade Inata/genética , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Análise por Conglomerados , Cruzamentos Genéticos , Primers do DNA , Fragaria/imunologia , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.
Assuntos
Proteínas de Ligação a DNA , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Modelos Genéticos , Plasmídeos/genética , Proteína Estafilocócica A/biossíntese , Simulação por Computador , Dano ao DNA , Desoxirribonuclease EcoRI/biossíntese , Desoxirribonuclease EcoRI/genética , Escherichia coli/classificação , Escherichia coli/metabolismo , Engenharia Genética/métodos , Cinética , Modelos Biológicos , Modelos Químicos , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Proteína Estafilocócica A/genética , Staphylococcus/genética , Transcrição Gênica , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
AIMS: Identification of two new plasmids in the multiple plasmid-containing strain V517 of Escherichia coli. METHOD AND RESULTS: By using an in-well mild cell lysis technique suitable for megaplasmids observation, two plasmids of 103.0 and 212.6 MDa were detected in the multiplasmid-containing E. coli V517. CONCLUSIONS: The two new megaplasmids that were completely overlooked when standard disruptive procedures were used, can now be added to the list of eight plasmids with molecular size from 1.36 to 35.84 MDa reported earlier. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding allows to use the strain V517 not only as a size reference of small and moderately large plasmids but as a size reference of megaplasmids as well.
Assuntos
Bacteriólise , Escherichia coli/genética , Escherichia coli/fisiologia , Técnicas Bacteriológicas , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Plasmídeos/fisiologiaRESUMO
Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganensis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dissipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability.
Assuntos
Actinomycetales/efeitos dos fármacos , Actinomycetales/patogenicidade , Antibacterianos/farmacologia , Actinomycetales/crescimento & desenvolvimento , Actinomycetales/metabolismo , Antibacterianos/isolamento & purificação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Frutas/microbiologia , Frutas/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Doenças das Plantas/microbiologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Sesquiterpenos , Terpenos , FitoalexinasRESUMO
The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/fisiologiaRESUMO
A mathematical model that includes biochemical interactions among the PTS system, phosphofructokinase (PFK), and pyruvate kinase (PK) is used to evaluate the dynamic behavior of the glycolytic pathway of Escherichia coli under steady-state conditions. The influence of ADP, phosphoenolpyruvate (PEP), and fructose-6-phosphate (F6P) on the dynamic regulation of this pathway is also analyzed. The model shows that the dynamic behavior of the system is affected significantly depending on whether ADP, PEP, or F6P is considered constant a steady state. Sustained oscillations are observed only when dADP/dt not equal 0 and completely suppressed if dADP/dt = 0 at any steady-state value. However, when PEP or F6P is constant, the system evolves toward the formation of stable limit cycles with periods ranging from 0.2 min to hours.
Assuntos
Difosfato de Adenosina/fisiologia , Escherichia coli/fisiologia , Trifosfato de Adenosina/fisiologia , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Glicólise , Cinética , Modelos Biológicos , Fosfoenolpiruvato/metabolismo , Fatores de TempoRESUMO
We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumán. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.
Assuntos
Azospirillum/isolamento & purificação , Produtos Agrícolas/microbiologia , Argentina , Raízes de Plantas/microbiologiaRESUMO
We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumán. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.
RESUMO
Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.