The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double-or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation. INTRODUCTIONAgrobacterium tumefaciens-mediated plant genetic transformation is an important experimental tool for investigation of various aspects of plant biology and for agricultural biotechnology. Development of this transformation technology represents the culmination of many decades of effort to improve tissue culture and plant genetic engineering techniques. Agrobacteriummediated transformation is based on a conjugative transfer-like process, which eventually takes the T-DNA to the host cell nucleus (Gelvin, 2000(Gelvin, , 2003a(Gelvin, , 2009Tzfira and Citovsky, 2003;Citovsky et al., 2007). After attachment of the bacterium to plant cells and induction of Agrobacterium virulence (vir) genes, a single-stranded DNA (the T-strand) is processed from the resident tumor-inducing or root-inducing plasmid and transported from the bacterium to the plant cell. In addition, a number of Virulence effector proteins, including VirD2 (attached to the T-strand), the single-strand DNA binding protein VirE2, plus VirE3, VirD5, and VirF are also transferred to the plant (Otten et al., 1984;Stahl et al., 1998;Vergunst et al., 2000Vergunst et al., , 2003Vergunst et al., , 2005Schrammeijer et al., 2003). These proteins are likely involved in protecting T-DNA from nuclease digestion, directing T-DNA to the nucleus, stripping proteins from the T-strand prior to integration, and integrating T-DNA into the plant genome. T-DNA integration occurs randomly into genomic DNA by the process of illegitimate recombination (...

BackgroundInternal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways.FindingsIndividual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed.Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus.ConclusionsThis study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.

Lactic acid bacteria are known for their biotechnological potential. In various regions of Ecuador numerous indigenous biological resources are largely undocumented. In this study, we evaluated the potential probiotic characteristics and antagonistic in vitro properties of some lactic acid bacteria from native niches of the subtropical rain forests of Ecuador. These isolates were identified according to their morphological properties, standard API50CH fermentation profile and RAPD-DNA polymorphism pattern. The selected isolates were further evaluated for their probiotic potential. The isolates grew at 15°C and 45°C, survived at a pH ranging from 2.5 to 4.5 in the presence of 0.3% bile (>90%) and grew under sodium chloride conditions. All selected isolates were sensitive to ampicillin, amoxicillin and cefuroxime and some showed resistance to gentamicin, kanamycin and tetracycline. Moreover, the agar well diffusion assay showed that the supernatant of each strain at pH 3.0 and pH 4.0, but not at pH 7.0 exhibited increased antimicrobial activity (inhibition zone >15mm) against two foodborne pathogens, Escherichia coli and Salmonella spp. To our knowledge, this is the first report describing the antagonistic activity against two foodborne pathogens and the probiotic in vitro potential of lactic acid bacteria isolated from native biota of Ecuador.

The use of peptides produced by lactic acid bacteria (LAB) as antimicrobial agents in food emerged from the increasing need of replacing chemicals with natural substances to ensure their safety and quality. A total of 30 LAB belonging to the genus Lactococcus sp. (10) and Enterococcus sp. (20) were isolated from native fruits of Ecuador subtropical rainforest. Among Lactococcus species, the isolates assigned Gt28, Gt29, and Ella8, identified as Lactococcus lactis subsp. lactis with 99% identity, showing highly inhibitory potential against four food pathogens were further characterized. The treatment of cell-free supernatant with proteolytic enzymes indicated the protein nature of released components, which displayed a broad antimicrobial activity against Gram-positive and -negative bacteria. Polymerase chain reaction analysis indicated the presence of lacticin 3147 gene in all isolates, lactococcin M gene in Gt28 and Gt29 but not in Ella8 and lactococcin A gene in Gt28 only. The antimicrobial activity was not linked to the presence of structural nisin gene as no amplification product was obtained. Treatment of Salmonella enterica ATCC 51741 and Escherichia coli ATCC 25922 at both vegetative and exponential phase of growth with the cell-free supernatant of Gt28 resulted in complete inactivation upon 3 h suggesting its bactericidal mode of action. An increment on inhibitory activity occurred when partial purified bacteriocin Gt28 was combined with ethylenediaminetetraacetic acid rather than bacteriocin only, indicating that the cells were sensitized in vitro by the chelator agent acting synergistically to induce the killing of pathogenic cells.

Tropical, wild-type fruits are considered biodiverse "hotspots" of microorganisms with possible functional characteristics to be investigated. In this study, several native lactic acid bacteria (LAB) of Ecuadorian Amazon showing highly inhibitory potential were identified and characterized. Based on carbohydrate fermentation profile and 16S rRNA gene sequencing, seven strains were assigned as Lactobacillus plantarum and one strain as Weissella confusa. Using agar-well diffusion method the active synthetized components released in the neutralized and hydroxide peroxide eliminated cell-free supernatant were inhibited by proteolytic enzymes, while the activity was maintained stable after the treatment with catalase, lysozyme, α-amylase and lipase suggesting their proteinaceous nature. The inhibitory activity was stimulated by acidic conditions, upon exposure to high heat and maintained stable at different ranges of sodium chloride (4-10%). The DNA sequencing analysis confirmed the presence of plw structural gene encoding for plantacirin W in the selected L. plantarum strains. Moreover, we showed that the active peptides of Cys5-4 strains contrast effectively, in a bactericidal manner, the growth of food borne E. coli UTNEc1 and Salmonella UTNSm2, with about tree fold reduction of viable counts at the early stage of the target cell growth. The results indicated that the bacteriocin produced by selected native lactic acid bacteria strains has elevated capacity to suppress several pathogenic microorganisms implying their potential as antimicrobial agents or food preservatives.