RESUMEN

Disease in agricultural field is a big problem that causes a massive loss in production. In this present investigation, we have reported a soil-borne bacterium Bacillus cereus IB311 which is antagonistic to plant pathogens (Pseudomonas syringae and Agrobacterium tumefaciens), and could make a substantial contribution to the prevention of plant diseases. To prove the practical application, the strain was directly applied in agricultural field. The results demonstrated that B. cereus IB311 has increased the production (20% and 26% in term of average pod number per plant, average seed number per pod, and seed yield per experimental plot) in ground nut (Arachis hypogaea var. Koushal, G201) and sesame (Sesamum indicum var. Kanak), respectively. To reduce the production cost, the biomass production was optimized through response surface methodology (RSM). Interactions of three variables (glucose, beef extract and inoculum) were studied using Central Composite Design. According to our analysis, optimum production of Bacillus cereus IB311 (5.383 µg/ mL) may be obtained at glucose 1.985%, beef extract 1.615% and inoculums size 0.757%. Therefore, we strongly believe that the application of this strain in agricultural field as bio-controlling agent will definitely enhance the production yield and will reduce the disease risk.

Asunto(s)

Inoculantes Agrícolas , Agrobacterium tumefaciens/crecimiento & desarrollo , Bacillus cereus , Agentes de Control Biológico , Biotecnología/métodos , Tumores de Planta/microbiología , Pseudomonas syringae/crecimiento & desarrollo , Biomasa , Productos Agrícolas/microbiología

Asunto(s)

Antibiosis , Bacillus/patogenicidad , Agrobacterium tumefaciens/crecimiento & desarrollo , Bacillus/fisiología , Recuento de Colonia Microbiana , Fusarium/crecimiento & desarrollo , Pectobacterium carotovorum/crecimiento & desarrollo , Pseudomonas fluorescens/crecimiento & desarrollo , Pseudomonas syringae/crecimiento & desarrollo , Xanthomonas campestris/crecimiento & desarrollo

RESUMEN

Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch.

Asunto(s)

Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/metabolismo , Sulfuro de Hidrógeno/metabolismo , Oxígeno/metabolismo , Plantas/microbiología , Factores de Transcripción/metabolismo , Xylella/metabolismo , Agrobacterium tumefaciens/crecimiento & desarrollo , Agrobacterium tumefaciens/fisiología , Animales , Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Secuencia Conservada , Cisteína , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Dioxigenasas/metabolismo , Disulfuros/química , Sulfuro de Hidrógeno/toxicidad , Ratones , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Operón/genética , Oxidación-Reducción , Estructura Secundaria de Proteína , Factores de Transcripción/química , Transcripción Genética , Xylella/crecimiento & desarrollo , Xylella/fisiología

RESUMEN

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.

Asunto(s)

Agrobacterium tumefaciens/genética , Blastocladiella/genética , Transformación Genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Blastocladiella/efectos de los fármacos , Blastocladiella/crecimiento & desarrollo , ADN Bacteriano/genética , Resistencia a Medicamentos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Higromicina B/farmacología , Microscopía Confocal , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa , Microbiología del Agua

RESUMEN

Guignardia citricarpa, the causal agent of Citrus Black Spot, was successfully transformed via Agrobacterium tumefaciens with cassettes for gfp and bar expression. Transformation is essential to understand the role of genes during interaction between plants and its pathogens. Using a binary plasmid vector based in the pPZP201BK, both germinated conidia and physically fragmented hyphae of G. citricarpa were transformed. Eight independent transformants of G. citricarpa resistant to ammonium glifosinate displayed GFP fluorescence. The majority (93.75%) of the G. citricarpa transformants was mitotically stable and contained a single T-DNA copy ectopically integrated to the chromosome. This is the first report of G. citricarpa transformation and will allow future work on virulence determinants of the fungus and possibly its control.

Asunto(s)

Agrobacterium tumefaciens/crecimiento & desarrollo , Agrobacterium tumefaciens/genética , Ascomicetos/genética , Técnicas de Transferencia de Gen , Transformación Genética , Ascomicetos/aislamiento & purificación , Citrus/microbiología , Hifa/genética , Enfermedades de las Plantas/microbiología , Plásmidos , Esporas Fúngicas/genética

RESUMEN

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L(-1)), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L(-1)) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.

Asunto(s)

Acinetobacter/crecimiento & desarrollo , Agrobacterium tumefaciens/crecimiento & desarrollo , Amidohidrolasas/metabolismo , Biopelículas/crecimiento & desarrollo , Reactores Biológicos/microbiología , Triazinas/metabolismo , Acinetobacter/clasificación , Acinetobacter/enzimología , Acinetobacter/genética , Agrobacterium tumefaciens/clasificación , Agrobacterium tumefaciens/enzimología , Agrobacterium tumefaciens/genética , Biodegradación Ambiental , Fuentes de Energía Bioeléctrica , Biotecnología/métodos , Células Inmovilizadas , Medios de Cultivo , ADN Bacteriano/genética , Herbicidas/metabolismo , Cinética , Reacción en Cadena de la Polimerasa , Técnica del ADN Polimorfo Amplificado Aleatorio

RESUMEN

Xylella fastidiosa is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in Xylella-plant interactions. Here we show that BigR (for "biofilm growth-associated repressor") is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta-lactamase-like hydrolase (BLH), is restricted to a few plant-associated bacteria, and thus, we sought to understand its regulation and function in X. fastidiosa and Agrobacterium tumefaciens. BigR binds to a palindromic AT-rich element (the BigR box) in the Xylella and Agrobacterium blh promoters and strongly represses the transcription of the operon in these cells. The BigR box overlaps with two alternative -10 regions identified in the blh promoters, and mutations in this box significantly affected transcription, indicating that BigR competes with the RNA polymerase for the same promoter site. Although BigR is similar to members of the ArsR/SmtB family of regulators, our data suggest that, in contrast to the initial prediction, it does not act as a metal sensor. Increased activity of the BigR operon was observed in both Xylella and Agrobacterium biofilms. In addition, an A. tumefaciens bigR mutant showed constitutive expression of operon genes and increased biofilm formation on glass surfaces and tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development.

Asunto(s)

Agrobacterium tumefaciens/crecimiento & desarrollo , Proteínas Bacterianas/fisiología , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/fisiología , Xylella/crecimiento & desarrollo , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Fusión Artificial Génica , Sitios de Unión/genética , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Secuencias Hélice-Giro-Hélice , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Operón , Regiones Promotoras Genéticas , Unión Proteica , Nicotiana/microbiología , Transcripción Genética , Xylella/genética

RESUMEN

Sunflower (Helianthus annuus L.) is considered one of the recalcitrant species in terms of transformation and regeneration. A routine transformation system of this crop requires competent cell cultures for efficient plant regeneration as well as an effective method for gene delivery. A transformation system was developed by an Agrobacterium tumefaciens-mediated method using split mature embryonic axis explants from the Ha89 genotype. Mean transformation efficiency obtained (measured as PCR+ plants/treated explants) varied from 1 to 5.2% depending on the use of the EHA105 or the C58 strain containing a plasmid with a gene of agronomic interest. The system developed has applicability to several Agrobacterium strains and plasmids with both reporter genes or genes of agronomic interest. Plants obtained with this protocol were confirmed by PCR and Southern blot. Stable inheritance of transgenes was successfully followed until generation T4 in several independent lines.

Asunto(s)

Agrobacterium tumefaciens/genética , Técnicas de Transferencia de Gen , Helianthus/genética , Plantas Modificadas Genéticamente/genética , Semillas/genética , Transformación Genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Southern Blotting/métodos , Helianthus/embriología , Helianthus/microbiología , Plantas Modificadas Genéticamente/embriología , Plantas Modificadas Genéticamente/microbiología , Reacción en Cadena de la Polimerasa/métodos , Regeneración/genética , Semillas/embriología , Semillas/microbiología , Análisis de Secuencia de ADN/métodos