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Sample records for agrobacterium mediated genetic

  1. Agrobacterium-mediated genetic transformation of Prunus salicina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report Agrobacterium tumefaciens-mediated transformation from hypocotyls slices of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supp...

  2. Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

    PubMed

    Mhatre, Minal

    2013-01-01

    Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

  3. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera

    PubMed Central

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  4. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

    PubMed Central

    Kwon, Tackmin

    2016-01-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  5. Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops.

    PubMed

    Singh, Roshan Kumar; Prasad, Manoj

    2016-05-01

    Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement.

  6. An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei.

    PubMed

    Kummasook, Aksarakorn; Cooper, Chester R; Vanittanakom, Nongnuch

    2010-12-01

    We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 10(4) conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.

  7. Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

    PubMed

    Tóth, Sándor; Kiss, Csaba; Scott, Peter; Kovács, Gabriella; Sorvari, Seppo; Toldi, Ottó

    2006-05-01

    In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R0 and R1 transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level.

  8. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

    PubMed Central

    Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

    2014-01-01

    Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important

  9. Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars.

    PubMed

    Arun, Muthukrishnan; Subramanyam, Kondeti; Mariashibu, Thankaraj Salammal; Theboral, Jeevaraj; Shivanandhan, Ganeshan; Manickavasagam, Markandan; Ganapathi, Andy

    2015-02-01

    Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.

  10. Genetic transformation of Metroxylon sagu (Rottb.) cultures via Agrobacterium-mediated and particle bombardment.

    PubMed

    Ibrahim, Evra Raunie; Hossain, Md Anowar; Roslan, Hairul Azman

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

  11. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

    PubMed Central

    Ibrahim, Evra Raunie

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  12. An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).

    PubMed

    Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath

    2013-09-01

    Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.

  13. High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

    PubMed Central

    Pathi, Krishna Mohan; Tula, Suresh; Tuteja, Narendra

    2013-01-01

    A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. PMID:23518589

  14. Agrobacterium-mediated sorghum transformation.

    PubMed

    Zhao, Z Y; Cai, T; Tagliani, L; Miller, M; Wang, N; Pang, H; Rudert, M; Schroeder, S; Hondred, D; Seltzer, J; Pierce, D

    2000-12-01

    Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

  15. A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.

    PubMed

    Li, Ying; Gao, Zhenrui; Piao, Chunlan; Lu, Kaiwen; Wang, Zhiping; Cui, Min-Long

    2014-02-01

    In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.

  16. Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana.

    PubMed

    dos Reis, Maria Cecília; Pelegrinelli Fungaro, Maria Helena; Delgado Duarte, Rubens Tadeu; Furlaneto, Luciana; Furlaneto, Marcia Cristina

    2004-08-01

    Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.

  17. An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

    PubMed

    Gupta, Vijayta; Ur Rahman, Laiq

    2015-07-01

    Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot

  18. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  19. Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

    PubMed Central

    2011-01-01

    Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated

  20. Barley Transformation Using Agrobacterium-Mediated Techniques

    NASA Astrophysics Data System (ADS)

    Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

    Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

  1. Agrobacterium-mediated transformation of Fusarium proliferatum.

    PubMed

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-06-03

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

  2. Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.

    PubMed

    Lee, Hyeyoung; Zhang, Zhanyuan J

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds.

  3. Agrobacterium: nature’s genetic engineer

    PubMed Central

    Nester, Eugene W.

    2015-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun’s old observations and also explain why Agrobacterium is nature’s genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

  4. Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.

    PubMed

    Krenek, Pavel; Samajova, Olga; Luptovciak, Ivan; Doskocilova, Anna; Komis, George; Samaj, Jozef

    2015-11-01

    Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.

  5. [Transformation of Didymella bryoniae mediated by Agrobacterium tumefaciens].

    PubMed

    Ren, Haiying; Fang, Li; Li, Gang; Ru, Shuijiang; Wang, Hanrong

    2010-06-01

    Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1 x 10(5) spores when 1 x 10(6) spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600 = 0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 microg/mL and selection medium contained 100 microg/mL of hygromycin B and 200 microg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.

  6. Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

    PubMed

    Han, J-S; Kim, C K; Park, S H; Hirschi, K D; Mok, I- G

    2005-03-01

    We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

  7. Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

    PubMed

    Sankari, Mohan; Hemachandran, Hridya; Anantharaman, Amirtha; Babu, Subramanian; Madrid, Renata Rivera; C, George Priya Doss; Fulzele, Devanand P; Siva, Ramamoorthy

    2016-07-01

    Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

  8. An efficient method of agrobacterium-mediated genetic transformation and regeneration in local Indian cultivar of groundnut (Arachis hypogaea) using grafting.

    PubMed

    Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

    2015-01-01

    Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69 ± 2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 ± 3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L(-1), respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65 ± 1.06 % regeneration, of which 3.82 ± 0.6 % explants were survived on hygromycin after selection. Finally, 14.58 ± 2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting.

  9. Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea.

    PubMed

    Chen, Liang; Wang, Qun; Chen, Hua; Sun, Gengwu; Liu, Huixiang; Wang, Hongkai

    2016-07-01

    Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10(5) protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis.

  10. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    PubMed Central

    2014-01-01

    Background The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. Conclusion The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome

  11. Optimization of Agrobacterium-Mediated Transformation in Soybean

    PubMed Central

    Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

    2017-01-01

    High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide

  12. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection.

    PubMed

    Hu, T; Metz, S; Chay, C; Zhou, H P; Biest, N; Chen, G; Cheng, M; Feng, X; Radionenko, M; Lu, F; Fry, J

    2003-06-01

    An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

  13. Genomic regions responsible for amenability to Agrobacterium-mediated transformation in barley

    PubMed Central

    Hisano, Hiroshi; Sato, Kazuhiro

    2016-01-01

    Different plant cultivars of the same genus and species can exhibit vastly different genetic transformation efficiencies. However, the genetic factors underlying these differences in transformation rate remain largely unknown. In barley, ‘Golden Promise’ is one of a few cultivars reliable for Agrobacterium-mediated transformation. By contrast, cultivar ‘Haruna Nijo’ is recalcitrant to genetic transformation. We identified genomic regions of barley important for successful transformation with Agrobacterium, utilizing the ‘Haruna Nijo’ × ‘Golden Promise’ F2 generation and genotyping by 124 genome-wide SNP markers. We observed significant segregation distortions of these markers from the expected 1:2:1 ratio toward the ‘Golden Promise’-type in regions of chromosomes 2H and 3H, indicating that the alleles of ‘Golden Promise’ in these regions might contribute to transformation efficiency. The same regions, which we termed Transformation Amenability (TFA) regions, were also conserved in transgenic F2 plants generated from a ‘Morex’ × ‘Golden Promise’ cross. The genomic regions identified herein likely include necessary factors for Agrobacterium-mediated transformation in barley. The potential to introduce these loci into any haplotype of barley opens the door to increasing the efficiency of transformation for target alleles into any haplotype of barley by the TFA-based methods proposed in this report. PMID:27874056

  14. Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation.

    PubMed

    Kishchenko, E M; Komarnitskii, I K; Kuchuk, N V

    2011-01-01

    Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).

  15. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

    PubMed

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants.

  16. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    PubMed

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  17. Development of Transgenic Papaya through Agrobacterium-Mediated Transformation

    PubMed Central

    Azad, Md. Abul Kalam; Rabbani, Md. Golam; Amin, Latifah; Sidik, Nik Marzuki

    2013-01-01

    Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

  18. Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds.

    PubMed

    Sujatha, M; Sailaja, M

    2005-03-01

    A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)). Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.

  19. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    PubMed

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  20. Agrobacterium delivers VirE2 protein into host cells via clathrin-mediated endocytosis

    PubMed Central

    Li, Xiaoyang; Pan, Shen Q.

    2017-01-01

    Agrobacterium tumefaciens can cause crown gall tumors on a wide range of host plants. As a natural genetic engineer, the bacterium can transfer both single-stranded DNA (ssDNA) [transferred DNA (T-DNA)] molecules and bacterial virulence proteins into various recipient cells. Among Agrobacterium-delivered proteins, VirE2 is an ssDNA binding protein that is involved in various steps of the transformation process. However, it is not clear how plant cells receive the T-DNA or protein molecules. Using a split–green fluorescent protein approach, we monitored the VirE2 delivery process inside plant cells in real time. We observed that A. tumefaciens delivered VirE2 from the bacterial lateral sides that were in close contact with plant membranes. VirE2 initially accumulated on plant cytoplasmic membranes at the entry points. VirE2-containing membranes were internalized through clathrin-mediated endocytosis to form endomembrane compartments. VirE2 colocalized with the early endosome marker SYP61 but not with the late endosome marker ARA6, suggesting that VirE2 escaped from early endosomes for subsequent trafficking inside the cells. Dual endocytic motifs at the carboxyl-terminal tail of VirE2 were involved in VirE2 internalization and could interact with the μ subunit of the plant clathrin-associated adaptor AP2 complex (AP2M). Both the VirE2 cargo motifs and AP2M were important for the transformation process. Because AP2-mediated endocytosis is well conserved, our data suggest that the A. tumefaciens pathogen hijacks conserved endocytic pathways to facilitate the delivery of virulence factors. This might be important for Agrobacterium to achieve both a wide host range and a high transformation efficiency. PMID:28345032

  1. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  2. Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

    PubMed

    Li, D D; Shi, W; Deng, X X

    2003-12-01

    Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

  3. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

    SciTech Connect

    Marton, L.

    1996-02-01

    Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

  4. Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors.

    PubMed

    Vega, Juan M; Yu, Weichang; Kennon, Angela R; Chen, Xinlu; Zhang, Zhanyuan J

    2008-02-01

    High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

  5. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

    PubMed Central

    Rana, Mohammad M.; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

    2016-01-01

    Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties. PMID:27428960

  6. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

    PubMed

    Michielse, C B; Ram, A F J; Hooykaas, P J J; Hondel, C A M J J van den

    2004-05-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.

  7. Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding.

    PubMed

    Zhang, Tian; Qi, Zhen; Wang, Yueyue; Zhang, Fangyuan; Li, Renyong; Yu, Qingsheng; Chen, Xiangbin; Wang, Huojun; Xiong, Xin; Tang, Kexuan

    2013-03-30

    Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/10(5)condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future.

  8. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation

    PubMed Central

    Liu, Yiming; Miao, Jiamin; Traore, Sy; Kong, Danyu; Liu, Yi; Zhang, Xunzhong; Nimchuk, Zachary L.; Liu, Zongrang; Zhao, Bingyu

    2016-01-01

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor. PMID:27833912

  9. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation.

    PubMed

    Liu, Yiming; Miao, Jiamin; Traore, Sy; Kong, Danyu; Liu, Yi; Zhang, Xunzhong; Nimchuk, Zachary L; Liu, Zongrang; Zhao, Bingyu

    2016-01-01

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.

  10. Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

    PubMed

    Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

    2015-12-14

    Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

  11. A high-efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2012-01-01

    Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

  12. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Brenna, Andrea; Montanini, Barbara; Muggiano, Eleonora; Proietto, Marco; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.

  13. Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi.

    PubMed

    Shao, Changwen; Yin, Youping; Qi, Zhaoran; Li, Ren; Song, Zhangyong; Li, Yan; Wang, Zhongkang

    2015-10-01

    An Agrobacterium-mediated genetic transformation system for the entomopathogenic fungus Nomuraea rileyi was established. Three binary T-DNA vectors, pPZP-Hph, pPZP-Hph-RNAi and pPZP-Hph-DsRed2, were constructed. The trpc promoter from Aspergillus nidulans was used as the cis-regulatory element to drive the expression of hygromycin phosphotransferase (hph) gene and DsRed2, which conferred the hygromycin B (Hyg B) resistance and red fluorescence visualization, respectively. The blastospores and conidia were used as the recipients. The blastospores' transformation efficiency reached ∼20-40 transformants per 10(6) blastospores, whereas the conidia were not transformed. Based on an analysis of five generations of subcultures, PCR and Southern blotting assays, the Ptrpc-hph cassette had integrated into the genomes of all transformants, which contained single copy of the hph gene and showed mitotic stability. Abundant altered morphologic phenotypes in colonies, blastospores and hyphae formations were observed in the arbitrary insertional mutants of N. rileyi, which made it possible to study the relationships between the functions and the interrupted genes over the whole genome. The transformation protocol will promote the functional characterization of genes, and the construction of genetically engineered strains of this important entomopathogenic fungus, and potentially of other similar fungal pathogens.

  14. [Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

    PubMed

    Zhou, Yue; Zhao, Xupeng; Wu, Xiuhua; Zhang, Yanling; Zhang, Lin; Luo, Keming; Tang, Shaohu

    2014-06-01

    Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

  15. Agrobacterium-mediated transformation of Euphorbia tirucalli callus.

    PubMed

    Uchida, Hidenobu; Yamashita, Hirofumi; Anai, Toyoaki; Muranaka, Toshiya; Ohyama, Kanji

    2010-01-01

    In order to establish a basis for transformation technology in the petroleum plant Euphorbia tirucalli, the callus of the plant was infected with Agrobacterium, washed with distilled water, sterilized with distilled water containing 100 mg/l of carbenicillin, selected on solidified B5 medium containing 13 mg/l of G418 and 100 mg/l of carbenicillin, and then on solidified B5 medium containing 25 mg/l of G418 and 100 mg/l of carbenicillin for the transgenic calli, and then the callus lines were subcultured successively on solidified B5 medium containing 50 mg/l of G418. We performed PCR analysis of sterilized G418-resistant callus line DNA and concluded that the gene introduced was integrated into the callus genome.

  16. Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants*#

    PubMed Central

    Li, Wen-xu; Wu, San-ling; Liu, Yan-hua; Jin, Gu-lei; Zhao, Hai-jun; Fan, Long-jiang; Shu, Qing-yao

    2016-01-01

    Agrobacterium-mediated transformation has been widely used in producing transgenic plants, and was recently used to generate “transgene-clean” targeted genomic modifications coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Although tremendous variation in morphological and agronomic traits, such as plant height, seed fertility, and grain size, was observed in transgenic plants, the underlying mechanisms are not yet well understood, and the types and frequency of genetic variation in transformed plants have not been fully disclosed. To reveal the genome-wide variation in transformed plants, we sequenced the genomes of five independent T0 rice plants using next-generation sequencing (NGS) techniques. Bioinformatics analyses followed by experimental validation revealed the following: (1) in addition to transfer-DNA (T-DNA) insertions, three transformed plants carried heritable plasmid backbone DNA of variable sizes (855–5216 bp) and in different configurations with the T-DNA insertions (linked or apart); (2) each transgenic plant contained an estimated 338–1774 independent genetic variations (single nucleotide variations (SNVs) or small insertion/deletions); and (3) 2–6 new Tos17 insertions were detected in each transformed plant, but no other transposable elements or bacterial genomic DNA. PMID:27921404

  17. Highly efficient transformation system for Malassezia furfur and Malassezia pachydermatis using Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Celis, A M; Vos, A M; Triana, S; Medina, C A; Escobar, N; Restrepo, S; Wösten, H A B; de Cock, H

    2017-03-01

    Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7days at 19°C and 24°C, respectively. Transformation efficiency was 0.75-1.5% for M. furfur and 0.6-7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.

  18. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  19. [Agrobacterium tumefaciens-mediated transformation of Aureobasidium pullulans and high-efficient screening for polymalic acid producing strain].

    PubMed

    Tu, Guangwei; Wang, Yongkang; Feng, Jun; Li, Xiaorong; Guo, Meijin; Zou, Xiang

    2015-07-01

    To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.

  20. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    PubMed

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

  1. Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

    PubMed

    Song, Guo-Qing; Sink, K C

    2004-12-01

    Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

  2. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    PubMed Central

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  3. An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants

    PubMed Central

    Nonaka, Satoko; Someya, Tatsuhiko; Zhou, Sha; Takayama, Mariko; Nakamura, Kouji; Ezura, Hiroshi

    2017-01-01

    Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato. PMID:28220841

  4. An efficient regeneration protocol for Agrobacterium-mediated transformation of melon (Cucumis melo L.).

    PubMed

    Zhang, H J; Gao, P; Wang, X Z; Luan, F S

    2014-01-08

    An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation, using cotyledon node zone-stem connection region of melon, has been developed. The new Agrobacterium-mediated transformation methodology, independent of organ culture, used the entire germinated seed as explants. The transformation system was maximized to maintain the integrity of melon itself, thus avoiding the limitations of traditional tissue culture methods. The transformation was carried out under a non-sterile environment. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed by PCR and Southern blot analyses. The transformation frequency based on the PCR was 13%. Transgenic melon plants were usually detected by PCR in less than 1 month after Agrobacterium inoculation, and seeds could be harvested in 3 months. The growth characteristics and morphology of the transgenic plants were identical to the untransformed wild-type plants. This method would be beneficial for facilitating the characteristics of gene functions and for boosting the manipulation of melon transformation for commercial purposes.

  5. Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill].

    PubMed

    Zeng, P; Vadnais, D A; Zhang, Z; Polacco, J C

    2004-02-01

    Modern genetic analysis and manipulation of soybean ( Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent. Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of L-cysteine for Williams 82. We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.

  6. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  7. Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.).

    PubMed

    Patil, Gunvant; Deokar, Amit; Jain, P K; Thengane, R J; Srinivasan, R

    2009-11-01

    To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l(-1) mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l(-1) mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.

  8. [Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

    PubMed

    Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

    2006-05-01

    The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement.

  9. Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.

    PubMed

    Gao, Xiquan; Shan, Libo

    2013-01-01

    Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.

  10. Transgenic plants from shoot apical meristems of Vitis vinifera L. "Thompson Seedless" via Agrobacterium-mediated transformation.

    PubMed

    Dutt, M; Li, Z T; Dhekney, S A; Gray, D J

    2007-12-01

    Shoot apical meristem explants of Vitis vinifera "Thompson Seedless" were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L(-1) in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L(-1) kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.

  11. Agrobacterium-mediated transformation of the endophytic fungus Acremonium implicatum associated with Brachiaria grasses.

    PubMed

    Abello, Javier; Kelemu, Segenet; García, Celsa

    2008-03-01

    Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophyte's plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.

  12. AGROBACTERIUM-MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

    PubMed

    Kathiresan, S; Chandrashekar, A; Ravishankar, G A; Sarada, R

    2009-06-01

    The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed β-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.

  13. Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

    PubMed

    Chattopadhyay, Tirthartha; Roy, Sheuli; Mitra, Adinpunya; Maiti, Mrinal K

    2011-04-01

    Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.

  14. Parameters influencing Agrobacterium-mediated transformation system in safflower genotypes AKS-207 and PKV Pink.

    PubMed

    Dhumale, Dipti Raghunath; Shingote, Prashant Raghunath; Dudhare, Mahendra Shankarrao; Jadhav, Pravin Vishwanath; Kale, Prashant Bhaskar

    2016-12-01

    Shoot regeneration in safflower (Carthamus tinctorius 'AKS 207' and 'PKV Pink') genetically transformed using Agrobacterium was used for assessing various constraints to the efficiency of transformation including infection period, virulence induction medium, co-cultivation period, bacterial titre, selection regime, and the natural phenolic compound acetosyringone. Transformation frequency was promising with 8-10-day-old cotyledonary leaf explants. Therefore, explants of that age cultured on Agrobacterium minimal medium (AB) containing 100 µM acetosyringone were infected with Agrobacterium (cell titre 0.5 OD600nm) for 15 min followed by 48 h of co-cultivation on kanamycin-enriched medium (50 mg/L). Transformation of the shoots was confirmed using β-glucuronidase (GUS) histochemical assay and polymerase chain reaction (PCR). With the transformation protocol thus optimized, the transformation frequency as determined using GUS assays was 54.0 % for AKS 207 and 47.6 % for PKV Pink. The corresponding figures using PCR were 27.0 and 33.3 %. The transformed shoots required 10-14 weeks of culture initiation but produced very few roots.

  15. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  16. Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

    PubMed Central

    Ziemienowicz, Alicja; Tinland, Bruno; Bryant, John; Gloeckler, Veronique; Hohn, Barbara

    2000-01-01

    Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP. PMID:10938108

  17. Transgenic grasspea (Lathyrus sativus L.): factors influencing agrobacterium-mediated transformation and regeneration.

    PubMed

    Barik, D P; Mohapatra, U; Chand, P K

    2005-11-01

    A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.

  18. Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.

    PubMed

    Lin, Xinping; Liu, Sasa; Bao, Ruiqi; Gao, Ning; Zhang, Sufang; Zhu, Rongqian; Zhao, Zongbao Kent

    2017-04-06

    Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.

  19. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    NASA Technical Reports Server (NTRS)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  20. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri.

    PubMed

    Rathod, Jayant Pralhad; Prakash, Gunjan; Pandit, Reena; Lali, Arvind M

    2013-11-01

    Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.

  1. Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation

    PubMed Central

    Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D.; Xia, Lan-Qin

    2016-01-01

    Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of “clean” GM wheat containing only the foreign genes of agronomic importance. PMID:27708648

  2. Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.

    PubMed

    Guo, M; Zhang, Y L; Meng, Z J; Jiang, J

    2012-03-16

    Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

  3. Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

    PubMed

    Alvarez, José M; Ordás, Ricardo J

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

  4. Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

    PubMed Central

    Alvarez, José M.; Ordás, Ricardo J.

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383

  5. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

    PubMed

    Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

    2015-01-01

    To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

  6. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    SciTech Connect

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  7. Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants.

    PubMed

    Petri, César; Wang, Hong; Alburquerque, Nuria; Faize, Mohamed; Burgos, Lorenzo

    2008-08-01

    A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.

  8. Efficient Agrobacterium tumefaciens-mediated transformation and regeneration of garlic (Allium sativum) immature leaf tissue.

    PubMed

    Kenel, Fernand; Eady, Colin; Brinch, Sheree

    2010-03-01

    Transgenic garlic (Allium sativum) plants have been recovered directly from immature leaf material by selective culture following Agrobacterium-mediated transformation. This method involved the use of a binary vector containing the mgfp-ER reporter gene and hpt selectable marker, and followed a similar protocol developed previously for the transformation of immature onion embryos. The choice of tissue and post-transformation selection procedure resulted in a large increase in recovery of transgenic plants compared with previously confirmed allium transformation protocols. The presence of transgenes in the genome of the plants was confirmed using Southern analysis. This improvement in frequency and the use of clonal commercial "Printanor" germplasm now makes possible the integration of useful agronomic and quality traits into this crop.

  9. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato.

    PubMed

    Sharma, Manoj K; Solanke, Amolkumar U; Jani, Dewal; Singh, Yogendra; Sharma, Arun K

    2009-09-01

    We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and beta-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

  10. Agrobacterium-mediated transformation of Malus robusta with tomato iron transporter gene.

    PubMed

    Qu, Shen-Chun; Huang, Xiao-De; Zhang, Zhen; Yao, Quan-Hong; Tao, Jian-Min; Qiao, Yu-Shan; Zhang, Jun-Yi

    2005-06-01

    The tomato iron transporter gene (LeIRT2) was introduced to Malus robusta Rehd. via Agrobacterium-mediated transformation to produce iron-deficiency tolerant apple rootstock. A total of 19 putative transformants were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. Two transgenic plants, one carrying one copy and the other three copies of the transgene, were hydroponically cultured to test their tolerance to iron-deficiency, which was found only in the transgenic plant with a single copy, which weighted 21%-4% greater than those of the control plants.

  11. Stable Recombinase-Mediated Cassette Exchange in Arabidopsis Using Agrobacterium tumefaciens1

    PubMed Central

    Louwerse, Jeanine D.; van Lier, Miranda C.M.; van der Steen, Dirk M.; de Vlaam, Clementine M.T.; Hooykaas, Paul J.J.; Vergunst, Annette C.

    2007-01-01

    Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation. PMID:17921337

  12. Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus.

    PubMed

    Clarke, Jihong Liu; Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W; Moe, Roar; Blystad, Dag-Ragnar

    2008-06-01

    Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

  13. Natural genetic transformation by agrobacterium rhizogenes . Annual flowering in two biennials, belgian endive and carrot

    PubMed

    Limami; Sun; Douat; Helgeson; Tepfer

    1998-10-01

    Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering. Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A. rhizogenes transformation. Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion. During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis. The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC (root locus) was the primary promoter of annualism and rolD caused extreme dwarfism. We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA.

  14. Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. x P. maximowiczii A. Henry.

    PubMed

    Yevtushenko, Dmytro P; Misra, Santosh

    2010-03-01

    Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. x P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the beta-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4-10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1-2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra x P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

  15. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    PubMed

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  16. A protocol for sonication-assisted Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explants.

    PubMed

    Klimek-Chodacka, Magdalena; Baranski, Rafal

    2014-01-01

    Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as a model system for studying plant metabolism and physiology, or can be utilized for the production of secondary metabolites. So far no efficient protocol of hairy root development in sugar beet has been publically released. In this work, two A. rhizogenes strains (A4T and LBA1334) carrying a binary vector pBIN-m-gfp5-ER or pCAMBIA1301 possessing gfp and uidA reporter genes were used to transform petiole explants of haploid and diploid sugar beet genotypes. Five treatment combinations of sonicated-assisted Agrobacterium-mediated transformation were compared. Hairy roots appeared on 0% to 54% of explants depending on the treatment combination used. The highest frequency was achieved when explants of a diploid genotype were sonicated for 15 s in the inoculum containing A. rhizogenes of OD600=0.5 and then co-cultured for three days. Using the same treatment combinations the explants of haploid genotypes developed hairy roots with the frequency ranging from 10% to 36%. Transformation efficiency was independent on the bacterial strain used. The results indicate that haploid sugar beet explants are amenable to transformation using A. rhizogenes, and that the efficiency of that process can be increased by applying short ultrasound treatment.

  17. Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.

    PubMed

    Luo, H; Hu, Q; Nelson, K; Longo, C; Kausch, A P; Chandlee, J M; Wipff, J K; Fricker, C R

    2004-04-01

    Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

  18. Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

    PubMed Central

    2011-01-01

    Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications. PMID:21595986

  19. Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Münch, Steffen; Ludwig, Nancy; Floss, Daniela S; Sugui, Janyce A; Koszucka, Anna M; Voll, Lars M; Sonnewald, Uwe; Deising, Holger B

    2011-01-01

    A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

  20. Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant.

    PubMed

    Pandey, Vibha; Misra, Pratibha; Chaturvedi, Pankaj; Mishra, Manoj K; Trivedi, Prabodh K; Tuli, Rakesh

    2010-02-01

    This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera--an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as compared to those from the in vitro-grown shoots. Second expanded leaf from the shoot tip gave the highest transient transformation efficiency. Selection of transgenic shoots was done in the presence of 50 mg l(-1) kanamycin. Polymerase chain reaction analysis of T(0) transgenic plants showed the presence of gusA and nptII genes. The expression of these transgenes in T(1) progeny was confirmed by RT-PCR. The integration of gusA gene was confirmed by Southern blot analysis. The transformation efficiency was found to be 1.67%.

  1. Establishment of an Agrobacterium-mediated Inoculation System for Cucumber Green Mottle Mosaic Virus

    PubMed Central

    Kang, Minji; Seo, Jang-Kyun; Song, Dami; Choi, Hong-Soo; Kim, Kook-Hyung

    2015-01-01

    The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor. PMID:26674677

  2. Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infiltration of tobacco leaves with a suspension of Agrobacterium tumefaciens harboring a binary plant expression plasmid provides a convenient method for laboratory scale protein production. When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana), diffic...

  3. Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

    PubMed

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-08-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

  4. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

    PubMed

    Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

    2017-04-01

    In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements.

  5. Transgene expression in tick cells using agrobacterium tumefaciens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ticks transmit infectious diseases to humans and other animals. Genetic manipulation of these arthropods would allow the development of alternative disease control strategies. Interestingly, Agrobacterium tumefaciens (At) mediated T-DNA transfer has been recently shown to promote the genetic modific...

  6. Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system.

    PubMed

    Gao, Zhensheng; Xie, Xueju; Ling, Yan; Muthukrishnan, Subbarat; Liang, George H

    2005-11-01

    A dual-marker plasmid containing the selectable marker gene, manA, and the reporter gene, sgfp, was used to transform immature sorghum embryos by employing an Agrobacterium-mediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi, was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1%-2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid. The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope. The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

  7. Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.

    PubMed

    Gasparis, Sebastian; Bregier, Cezary; Orczyk, Waclaw; Nadolska-Orczyk, Anna

    2008-11-01

    This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes.

  8. Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression.

    PubMed

    Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

    2010-12-01

    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.

  9. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

    PubMed

    Rakosy-Tican, Elena; Aurori, Cristian M; Dijkstra, Camelia; Thieme, Ramona; Aurori, Adriana; Davey, Michael R

    2007-05-01

    Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

  10. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2009-04-01

    Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

  11. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

  12. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  13. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  14. Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops.

    PubMed

    Bosmans, Lien; Álvarez-Pérez, Sergio; Moerkens, Rob; Wittemans, Lieve; Van Calenberge, Bart; Kerckhove, Stefan Van; Paeleman, Anneleen; De Mot, René; Rediers, Hans; Lievens, Bart

    2015-08-01

    Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium.

  15. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

    PubMed

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

  16. A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

    PubMed Central

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

  17. Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...

  18. Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

  19. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

  20. A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.

    PubMed

    Păcurar, Daniel Ioan; Thordal-Christensen, Hans; Nielsen, Klaus Kristian; Lenk, Ingo

    2008-10-01

    In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.

  1. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene

    PubMed Central

    2012-01-01

    Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1 along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated. PMID:22691292

  2. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    NASA Technical Reports Server (NTRS)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  3. Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that underpin pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transform...

  4. Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative n...

  5. Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

    NASA Astrophysics Data System (ADS)

    Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

    This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

  6. Cell wall biochemical alterations during Agrobacterium-mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco.

    PubMed

    Le Mauff, François; Loutelier-Bourhis, Corinne; Bardor, Muriel; Berard, Caroline; Doucet, Alain; D'Aoust, Marc-André; Vezina, Louis-Philippe; Driouich, Azeddine; Couture, Manon M-J; Lerouge, Patrice

    2017-03-01

    Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.

  7. Agrobacterium tumefaciens-mediated transformation of the causative agent of Valsa canker of apple tree Valsa mali var. mali.

    PubMed

    Hu, Yang; Dai, Qingqing; Liu, Yangyang; Yang, Zhe; Song, Na; Gao, Xiaoning; Voegele, Ralf Thomas; Kang, Zhensheng; Huang, Lili

    2014-06-01

    Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10(6) conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.

  8. Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

    PubMed

    Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

    2017-02-01

    An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

  9. Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

    PubMed

    Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F

    2014-02-21

    Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

  10. Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Camilo, César M

    2011-08-01

    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.

  11. Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos.

    PubMed

    Jube, Sandro; Borthakur, Dulal

    2009-01-01

    The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was con- firmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol.

  12. Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.

    PubMed

    Taniguchi, T; Kurita, M; Ohmiya, Y; Kondo, T

    2005-03-01

    A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis.

  13. High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

    PubMed

    Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

    2011-09-01

    To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

  14. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

    PubMed

    Ogita, Shinjiro; Usui, Miki; Shibutani, Nanae; Kato, Yasuo

    2009-07-01

    A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

  15. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    PubMed

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  16. Agrobacterium tumefaciens-mediated transformation of Valsa mali: an efficient tool for random insertion mutagenesis.

    PubMed

    Wang, Caixia; Guan, Xiangnan; Wang, Hanyan; Li, Guifang; Dong, Xiangli; Wang, Guoping; Li, Baohua

    2013-01-01

    Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT), with the optimal transformation conditions as follows: 10(6)/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200  μ M acetosyringone (AS) in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 10(6) recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels.

  17. Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters.

    PubMed

    Lee, M H; Yoon, E S; Jeong, J H; Choi, Y E

    2004-06-01

    Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5+/-3.5%) as compared to stem (32.7+/-4.8%) or cotyledon (16.2+/-5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5+/-9.8%) than that of non-transformed roots (31.7 +/-9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.

  18. Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation.

    PubMed

    Matvieieva, N A; Shakhovsky, A M; Belokurova, V B; Drobot, K O

    2016-05-18

    An efficient and rapid protocol for the establishment of Artemisia tilesii "hairy" root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5-6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii "hairy" root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA-up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 μl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 μl/L concentration led to 5.46-fold mass increase in only one "hairy" root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.

  19. Nitrite-mediated synthesis of chiral epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1.

    PubMed

    Jin, Huo-Xi; Hu, Zhong-Ce; Liu, Zhi-Qiang; Zheng, Yu-Guo

    2012-01-01

    In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni-nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral epichlorohydrin in the presence of NO₂⁻. The optimal pH, temperature, and NO₂⁻ concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)-epichlorohydrin are 714.3 µmol min⁻¹ mg⁻¹ and 17.2 mM, respectively, whereas those for (R)-epichlorohydrin are 166.8 µmol min⁻¹ mg⁻¹ and 29.0 mM, respectively. Under optimal conditions, (R)-epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)-epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3-dichloro-2-propanol by the ring opening of racemic epichlorohydrin in the presence of NO₂⁻ after the ring closure of 1,3-dichloro-2-propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of epichlorohydrin with NO₂⁻ and synthesis of chiral epichlorohydrin with 99% enantiomeric excess from 1,3-dichloro-2-propanol by combining ring closure of 1,3-dichloro-2-propanol and ring opening of racemic epichlorohydrin.

  20. Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat.

    PubMed

    Przetakiewicz, Anna; Karaś, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2004-01-01

    Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.

  1. Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.

    PubMed

    Zhang, Yan-Jun; Zhao, Jin-Jin; Xie, Ming; Peng, De-Liang

    2014-10-01

    Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain.

  2. Development of Marker-Free Insect-Resistant Indica Rice by Agrobacterium tumefaciens-Mediated Co-transformation.

    PubMed

    Ling, Fei; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2016-01-01

    Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A(∗) containing a synthetic cry2A(∗) gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt) were introduced into Minghui86 (Oryza sativa L. ssp. indica), an elite indica restorer line. Two independent transformants containing both the cry2A(∗) gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas) and rice leaf folder (Cnaphalocrocis medinalis). The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690). These results indicate that the marker-free transgenic rice has the potential for commercial production.

  3. Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation.

    PubMed

    Han, Yun-Jeong; Kim, Yong-Min; Lee, Jee-Yeon; Kim, Soo Jung; Cho, Kyu-Chang; Chandrasekhar, Thummala; Song, Pill-Soon; Woo, Young-Min; Kim, Jeong-Il

    2009-03-01

    Purple-colored transgenic creeping bentgrass (Agrostis stolonifera L.) plants were developed for ornamental purpose by means of Agrobacterium-mediated transformation. Embryogenic creeping bentgrass calli were transformed with the pCAMBIA 3301 vector harboring maize (Zea mays) flavonoid/anthocyanin biosynthetic pathway transcription factor genes, Lc (Leaf color) and Pl (Purple leaf), individually and in combination, and three types of putative transgenic plants (Lc, Pl, and Lc + Pl) were generated. Genomic integration and expression of the transgenes were confirmed by Southern and northern blot analyses, respectively. The transgenic creeping bentgrass plants expressing both Lc and Pl genes were entirely purple, whereas those expressing Pl alone had purple stems and those expressing Lc alone lacked purple pigmentation in adult plants. The anthocyanin content was estimated in all the three types of transgenic plant and correlated well with the degree of purple coloration observed. These results suggest that both Lc and Pl genes are necessary and sufficient to confer purple coloration to creeping bentgrass.

  4. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    PubMed

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  5. Development of Marker-Free Insect-Resistant Indica Rice by Agrobacterium tumefaciens-Mediated Co-transformation

    PubMed Central

    Ling, Fei; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2016-01-01

    Agrobacterium-mediated co-transformation is an efficient strategy to generate marker-free transgenic plants. In this study, the vectors pMF-2A∗ containing a synthetic cry2A∗ gene driven by maize ubiquitin promoter and pCAMBIA1301 harboring hygromycin phosphotransferase gene (hpt) were introduced into Minghui86 (Oryza sativa L. ssp. indica), an elite indica restorer line. Two independent transformants containing both the cry2A∗ gene and hpt gene were regenerated. Several homozygous marker-free transgenic progenies were derived from family 2AH2, and three of them were selected for further insect bioassay in the laboratory and field. Insect-resistance assays revealed that all the three transgenic lines were highly resistant to striped stem borer (Chilo suppressalis), yellow stem borer (Tryporyza incertulas) and rice leaf folder (Cnaphalocrocis medinalis). The measurement of Cry2A protein concentration showed that Cry2A protein was stably expressed in leaves and stems of homozygous transgenic lines and their hybrids. The yields of the marker-free homozygous transgenic lines and their hybrids were not significantly different from those of their corresponding controls. Furthermore, the results of flanking sequence isolation showed that the T-DNA in line 8-30 was integrated into the intergenic region of chromosome 2 (between Os02g43680 and Os02g43690). These results indicate that the marker-free transgenic rice has the potential for commercial production. PMID:27833629

  6. Proteomics and Genetics for Identification of a Bacterial Antimonite Oxidase in Agrobacterium tumefaciens.

    PubMed

    Li, Jingxin; Wang, Qian; Li, Mingshun; Yang, Birong; Shi, Manman; Guo, Wei; McDermott, Timothy R; Rensing, Christopher; Wang, Gejiao

    2015-05-19

    Antimony (Sb) and its compounds are listed by the United States Environmental Protection Agency (USEPA, 1979) and the European Union (CEC, 1976) as a priority pollutant. Microbial redox transformations are presumed to be an important part of antimony cycling in nature; however, regulation of these processes and the enzymology involved are unknown. In this study, comparative proteomics and reverse transcriptase-PCR analysis of Sb(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 revealed an oxidoreductase (anoA) is widely distributed in microorganisms, including at least some documented to be able to oxidize Sb(III). Deletion of the anoA gene reduced Sb(III) resistance and decreased Sb(III) oxidation by ∼27%, whereas the anoA complemented strain was similar to the wild type GW4 and a GW4 anoA overexpressing strain increased Sb(III) oxidation by ∼34%. Addition of Sb(III) up-regulated anoA expression and cloning anoA to Escherichia coli demonstrated direct transferability of this activity. A His-tag purified AnoA was found to require NADP(+) as cofactor, and exhibited a K(m) for Sb(III) of 64 ± 10 μM and a V(max) of 150 ± 7 nmol min(-1) mg(-1). This study contributes important initial steps toward a mechanistic understanding of microbe-antimony interactions and enhances our understanding of how microorganisms participate in antimony biogeochemical cycling in nature.

  7. An efficient protocol for genetic transformation of watercress (Nasturtium officinale) using Agrobacterium rhizogenes.

    PubMed

    Park, Nam Il; Kim, Jae Kwang; Park, Woo Tae; Cho, Jin Woong; Lim, Yong Pyo; Park, Sang Un

    2011-11-01

    Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01-0.02 μmol/g of DW) and 4-methoxyglucobrassicin (0.06-0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06-0.21 μmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).

  8. Reproduction of Meloidogyne javanica on Plant Roots Genetically Transformed by Agrobacterium rhizogenes.

    PubMed

    Verdejo, S; Jaffee, B A; Mankau, R

    1988-10-01

    Reproduction of Meloidogyne javanica was compared on several Agrobacterium rhizogenes-transformed root cultures under monoxenic conditions. M. javanica reproduced on all transformed roots tested; however, more females and eggs were obtained on potato and South Australian Early Dwarf Red tomato than on bindweed, Tropic tomato, lima bean, or carrot. Roots that grew at moderate rates into the agar and produced many secondary roots supported the highest reproduction. Numbers of females produced in cultures of transformed potato roots increased with increasing nematode inoculum levels, whether inoculum was dispersed eggs or juveniles. Females appeared smaller, produced fewer eggs, and were found in coalesced galls at the higher inoculum levels. The ratio between the final and initial population decreased sharply as the juvenile inoculum increased. The second-stage juvenile was preferred to dispersed eggs or egg masses for inoculation of tissue culture systems because quantity and viability of inoculum were easily assessed. Meloidogyne javanica reared on transformed root cultures were able to complete their life cycles on new transformed root cultures or greenhouse tomato plants.

  9. Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium.

    PubMed

    Khanna, H K; Daggard, G E

    2003-01-01

    Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.

  10. Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii.

    PubMed

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J

    2008-05-01

    Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion.

  11. Construction of an engineering strain producing high yields of α-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger.

    PubMed

    Li, Ming; Zhou, Liying; Liu, Meng; Huang, Yunyan; Sun, Xin; Lu, Fuping

    2013-01-01

    In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was used in breeding industrial strains for the purpose of improving α-transglucosidase production. Firstly, an efficient ATMT system for Asperillus niger was established by optimization of several influencing factors, in which transformation efficiency was improved up to 14-fold compared with the initial conditions. Furthermore, binary vector pBI-Glu containing an α-transglucosidase expression cassette was constructed and transferred into Agrobacterium tumefaciens LBA4404 in order to infect A. niger. By the efficient ATMT method, the gene for α-transglucosidase, driven by strong promoter PglaA (the glucoamylase gene promoter), had a high expression level in A. niger A-8 (25.02 U/mL). The optimized ATMT system was found to be effective and suitable for A. niger, and should be a useful tool for studying the function of A. niger genes and for industrial breeding of this strain.

  12. Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens.

    PubMed

    Park, Yong-Soon; Ryu, Choong-Min

    2015-01-01

    Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria.

  13. Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease.

    PubMed

    Elayabalan, Sivalingam; Kalaiponmani, Kalaimughilan; Subramaniam, Sreeramanan; Selvarajan, Ramasamy; Panchanathan, Radha; Muthuvelayoutham, Ramlatha; Kumar, Krish K; Balasubramanian, Ponnuswami

    2013-04-01

    One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana.

  14. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

    PubMed

    Wang, Ke; Liu, Huiyun; Du, Lipu; Ye, Xingguo

    2016-11-10

    Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker-free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium-mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker-free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T-DNA regions. The average co-integration frequency of the gus and the bar genes located on the two independent T-DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker-free plants was related to the number of bar gene copies integrated in the genome. Marker-free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.

  15. Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents

    PubMed Central

    Huang, Bing; Lin, Huanjie; Yan, Chuanyan; Qiu, Hongyan; Qiu, Lipeng; Yu, Rongmin

    2014-01-01

    Background: Polygonum multiflorum is an important medicinal plant. Hairy roots systems obtained by transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes can produce valuable biological active substances, which have immense potential in the pharmaceutical industry. Objective: To optimize the inductive and cultural conditions of P. multiflorum hairy roots and to identify the major active secondary metabolites in hairy roots. Materials and Methods: P. multiflorum hairy root were mediated with A. rhizogenes R1601 to induce hairy roots. Four combinations, including Murashige–Skoog (MS), 1/2 MS, B5, and White, were investigated to optimize the culture medium. MS medium was selected for the growth measurement. The qualitative and quantitative determinations of free anthraquinone in hairy roots were compared with the calli and aseptic plantlets using high-performance liquid chromatography. Results: The inductive rates of hairy roots by leaves were higher than for any other explants. The presence of agropine in the P. multiflorum hairy roots confirmed that they were indeed transgenic. MS medium was the most suitable of the four media for hairy root growth. Meanwhile, the growth kinetics and nutrient consumption results showed that the hairy roots displayed a sigmoidal growth curve and that their optimal inoculation time was 18-21 days. The determination of the anthraquinone constituents indicated that the rhein content of the hairy roots reached 2.495 μg g−1 and was 2.55-fold higher than that of natural plants. Conclusion: Transgenic hairy roots of P. multiflorum could be one of the most potent materials for industrial-scale production of bioactive anthraquinone constituents. PMID:24696550

  16. Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).

    PubMed

    Zhang, C L; Chen, D F; McCormac, A C; Scott, N W; Elliott, M C; Slater, A

    2001-02-01

    Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2-5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

  17. Highly efficient transformation and plant regeneration of tall fescue mediated by Agrobacterium tumefaciens.

    PubMed

    Hu, Zhang-Hua; Chen, Jin-Qing; Wu, Guan-Ting; Jin, Wei; Lang, Chun-Xiu; Huang, Rui-Zhi; Wang, Fu-Lin; Liu, Zhi-Hong; Chen, Xiao-Yun

    2005-04-01

    An efficient and reproducible system has been developed for the production of transgenic plants in tall fescue (Festuca arundinacea Schreb.) using A. tumefaciens-mediated transformation. Two-months-old suspension cultures served as excellent explants for transformation. The explants were inoculated with an A. tumefaciens strain EHA105 carrying a plasmid pDBA121 containing genes for hygromycin phosphotransferase (hpt) and phosphinotricin acetyltransferase (bar). The commercial herbicide Basta was used as a selective agent. Inclusion of acetosyringone (ACS) 20 mg/L in the co-culture medium led to an increase in transformation efficiency. The efficiency of transformation was highly dependent on the genotype, the explant, the culture medium and selective agent used. Tall fescue transformation efficiency is 2.85-10.9 plants per gram fresh weight (FW) of suspension cultures. This is much higher than the corresponding values reported before (2-5 plants). So far more than 300 transgenic plants have been obtained, the fertility of some transgenic plants had decreased. Stable integration and high expression of the transgenes were confirmed by PCR analysis and Southern blot hybridization or herbicide Basta spraying test.

  18. Excursion-Set-Mediated Genetic Algorithm

    NASA Technical Reports Server (NTRS)

    Noever, David; Baskaran, Subbiah

    1995-01-01

    Excursion-set-mediated genetic algorithm (ESMGA) is embodiment of method of searching for and optimizing computerized mathematical models. Incorporates powerful search and optimization techniques based on concepts analogous to natural selection and laws of genetics. In comparison with other genetic algorithms, this one achieves stronger condition for implicit parallelism. Includes three stages of operations in each cycle, analogous to biological generation.

  19. Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

    PubMed

    Yadav, Reena; Mehrotra, Meenakshi; Singh, Aditya K; Niranjan, Abhishek; Singh, Rani; Sanyal, Indraneel; Lehri, Alok; Pande, Veena; Amla, D V

    2017-01-01

    Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

  20. Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture.

    PubMed

    O'Neill, Kristin M; Larsen, Jeffrey S; Curtis, Wayne R

    2008-01-01

    The reporter gene beta-glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through co-culture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.

  1. Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).

    PubMed

    Subramanyam, Kondeti; Rajesh, Manoharan; Jaganath, Balusamy; Vasuki, Amirthalingam; Theboral, Jeevaraj; Elayaraja, Dhandapani; Karthik, Sivabalan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-09-01

    An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 μM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).

  2. A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant.

    PubMed

    Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

    2014-09-01

    In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.

  3. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

    PubMed

    Nyaboga, Evans N; Njiru, Joshua M; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.

  4. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

    PubMed Central

    Nyaboga, Evans N.; Njiru, Joshua M.; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  5. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brac...

  6. Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae.

    PubMed

    Dobinson, Katherine F; Grant, Sandra J; Kang, Seogchan

    2004-02-01

    A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae. The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids. The gene is present in V. dahliae isolates from different host plants and in V. albo-atrum; weakly hybridizing sequences are present in V. tricorpus. VTP1 cDNA sequences were identified in a sequence tag analysis of genes expressed under growth conditions that promote microsclerotia development. Replacement of the gene, by Agrobacterium tumefaciens-mediated transformation (ATMT), with a mutant allele construct did not noticeably alter either pathogenicity or growth in culture. Searches of expressed sequence tag databases showed that, in addition to the VTP1 gene, V. dahliae contains two genes encoding subtilisin-like proteases similar to those produced by pathogenic Aspergillus spp. This is the first description of the application of ATMT to the molecular analysis of phytopathogenic Verticillium spp.

  7. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

    PubMed Central

    Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2015-01-01

    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

  8. Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

    PubMed

    Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

    2015-11-01

    An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

  9. The Agrobacterium Ti Plasmids.

    PubMed

    Christie, Peter J; Gordon, Jay E

    2014-12-01

    Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing or Ti plasmid to susceptible plant cells. A. tumefaciens belongs to the class Alphaproteobacteria, whose members include other plant pathogens (Agrobacterium rhizogenes), plant and insect symbionts (Rhizobium spp. and Wolbachia spp., respectively), human pathogens (Brucella spp., Bartonella spp., Rickettsia spp.), and nonpathogens (Caulobacter crescentus, Rhodobacter sphaeroides). Many species of Alphaproteobacteria carry large plasmids ranging in size from ∼100 kb to nearly 2 Mb. These large replicons typically code for functions essential for cell physiology, pathogenesis, or symbiosis. Most of these elements rely on a conserved gene cassette termed repABC for replication and partitioning, and maintenance at only one or a few copies per cell. The subject of this review is the ∼200-kb Ti plasmids carried by infectious strains of A. tumefaciens. We will summarize the features of this plasmid as a representative of the repABC family of megaplasmids. We will also describe novel features of this plasmid that enable A. tumefaciens cells to incite tumor formation in plants, sense and respond to an array of plant host and bacterial signal molecules, and maintain and disseminate the plasmid among populations of agrobacteria. At the end of this review, we will describe how this natural genetic engineer has been adapted to spawn an entire industry of plant biotechnology and review its potential for use in future therapeutic applications of plant and nonplant species.

  10. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.

    PubMed

    Ma, Zhiying; Liu, Jianfeng; Wang, Xingfen

    2013-01-01

    Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method.

  11. Fundamental discoveries and simple recombination between circular plasmid DNAs led to widespread use of Agrobacterium tumefaciens as a generalized vector for plant genetic engineering.

    PubMed

    Zambryski, Patricia

    2013-01-01

    Fundamental research aimed to determine the limits of the Agrobacterium transfer DNA (T-DNA) element that stably inserted into plant nuclear DNA to cause crown gall tumor formation. The T-DNA borders were discovered to be exceedingly precise, revealing that T-DNA insertion into the plant genome was reproducible and exact. Deletion of the internal regions of the T-DNA, to remove the tumor forming genes, while retaining the T-DNA borders, resulted again in efficient DNA transfer to plant cells, but now such cells were capable of completely normal growth and differentiation. Thus, the internal region of the T-DNA was not needed for DNA transfer, and one could envisage insertion of any DNA of interest in between the T-DNA borders. Thus began plant genetic engineering.

  12. Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper).

    PubMed

    Sainger, Manish; Chaudhary, Darshna; Dahiya, Savita; Jaiwal, Ranjana; Jaiwal, Pawan K

    2015-10-01

    An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their

  13. IMPROVING PLANT GENETIC ENGINEERING BY MANIPULATING THE HOST. (R829479C001)

    EPA Science Inventory

    Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to ...

  14. Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

    PubMed

    Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

    2007-10-01

    A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

  15. Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation.

    PubMed

    Tanaka, Hidenori; Toyama, Jun; Hashiguchi, Masatsugu; Kutsuna, Yasuyo; Tsuruta, Shin-ichi; Akashi, Ryo; Hoffmann, Franz

    2008-08-25

    Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

  16. Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay.

    PubMed

    Banu, Sufara Akhter; Huda, Kazi Md Kamrul; Tuteja, Narendra

    2014-04-30

    Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.

  17. Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.

    PubMed

    Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

    2014-05-01

    Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.

  18. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.

    PubMed

    Maruthachalam, K; Klosterman, S J; Kang, S; Hayes, R J; Subbarao, K V

    2011-11-01

    Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.

  19. Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols.

    PubMed

    Ramesh, Sunita A; Kaiser, Brent N; Franks, Tricia; Collins, Graham; Sedgley, Margaret

    2006-08-01

    A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

  20. Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

    PubMed Central

    Manfroi, Ernandes; Yamazaki-Lau, Elene; Grando, Magali F.; Roesler, Eduardo A.

    2015-01-01

    Abstract Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future. PMID:26537604

  1. Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2015-11-20

    During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

  2. The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

    PubMed

    Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2012-02-01

    The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

  3. Horizontal gene transfer from Agrobacterium to plants

    PubMed Central

    Matveeva, Tatiana V.; Lutova, Ludmila A.

    2014-01-01

    Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

  4. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  5. Agrobacterium-mediated transformation of potato using PLRV-rep and PVY CP genes and assessment of replicase mediated resistance against natural infection of PLRV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Replicase-and coat protein gene-mediated resistances against potato leafroll virus (PLRV) and potato virus Y (PVY), respectively, demonstrated to be an effective way of protecting potato against two major virus problems (PLRV & PVY) world-wide. Potato cultivar Desiree was transformed using Agrobacte...

  6. Agrobacterium tumefaciens as an agent of disease.

    PubMed

    Escobar, Matthew A; Dandekar, Abhaya M

    2003-08-01

    Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer. Since this discovery, A. tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange. It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology. These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research. Now, the diverse areas of A. tumefaciens research are again converging because new discoveries in transformation biology and the use of A. tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.

  7. Odyssey of agrobacterium T-DNA.

    PubMed

    Ziemienowicz, A

    2001-01-01

    Agrobacterium tumefaciens, a plant pathogen, is characterized by the unique feature of interkingdom DNA transfer. This soil bacterium is able to transfer a fragment of its DNA, called T-DNA (transferred DNA), to the plant cell where T-DNA is integrated into the plant genome leading to "genetic colonization" of the host. The fate of T-DNA, its processing, transfer and integration, resembles the journey of Odysseus, although our hero returns from its long trip in a slightly modified form.

  8. Development of an efficient agrobacterium-mediated gene targeting system for rice and analysis of rice knockouts lacking granule-bound starch synthase (Waxy) and β1,2-xylosyltransferase.

    PubMed

    Ozawa, Kenjirou; Wakasa, Yuhya; Ogo, Yuko; Matsuo, Kouki; Kawahigashi, Hiroyuki; Takaiwa, Fumio

    2012-04-01

    We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

  9. Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

    PubMed Central

    Izawati, Abang Masli Dayang; Masani, Mat Yunus Abdul; Ismanizan, Ismail; Parveez, Ghulam Kadir Ahmad

    2015-01-01

    DOGR1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l-1 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm. PMID:26442041

  10. Transformation of medicinal plants using Agrobacterium tumefaciens.

    PubMed

    Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata

    2016-12-20

    For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

  11. Transformation of oil palm using Agrobacterium tumefaciens.

    PubMed

    Izawati, Abang Masli Dayang; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul

    2012-01-01

    Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.

  12. Recombinant synthesis of hyaluronan by Agrobacterium sp.

    PubMed

    Mao, Zichao; Chen, Rachel Ruizhen

    2007-01-01

    Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications.

  13. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells

    PubMed Central

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-01-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20–25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

  14. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

    PubMed

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-02-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.

  15. Induction of Pseudoactinorhizae by the Plant Pathogen Agrobacterium rhizogenes.

    PubMed

    Berg, R H; Liu, L; Dawson, J O; Savka, M A; Farrand, S K

    1992-02-01

    Infection of Elaeagnus angustifolia cotyledonary wounds by Agrobacterium rhizogenes strain NCPPB 2659 resulted in the formation of pseudoactinorhizae on roots differentiated from callus. These pseudoactinorhizal root nodules were anatomically indistinguishable from the actinorhizae induced by the plant's microsymbiont Frankia. This unusual hairy root phenotype provides support for the concept that the genetic program for actinorhiza morphogenesis resides in the plant's genome.

  16. Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants.

    PubMed

    Bosselut, Nathalie; Van Ghelder, Cyril; Claverie, Michel; Voisin, Roger; Onesto, Jean-Paul; Rosso, Marie-Noëlle; Esmenjaud, Daniel

    2011-07-01

    Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum × almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.

  17. Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

    PubMed

    Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita

    2011-05-01

    We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.

  18. [Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

    PubMed

    Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

    2014-01-01

    To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

  19. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum

    PubMed Central

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; Vogel, John P.; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation. PMID:27252729

  20. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    DOE PAGES

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; ...

    2016-05-24

    In this study, the genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This studymore » demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.« less

  1. Religiousness, antisocial behavior, and altruism: genetic and environmental mediation.

    PubMed

    Koenig, Laura B; McGue, Matt; Krueger, Robert F; Bouchard, Thomas J

    2007-04-01

    Although religiousness is considered a protective factor against antisocial behaviors and a positive influence on prosocial behaviors, it remains unclear whether these associations are primarily genetically or environmentally mediated. In order to investigate this question, religiousness, antisocial behavior, and altruistic behavior were assessed by self-report in a sample of adult male twins (165 MZ and 100 DZ full pairs, mean age of 33 years). Religiousness, both retrospective and current, was shown to be modestly negatively correlated with antisocial behavior and modestly positively correlated with altruistic behavior. Joint biometric analyses of religiousness and antisocial behavior or altruistic behavior were completed. The relationship between religiousness and antisocial behavior was due to both genetic and shared environmental effects. Altruistic behavior also shared most all of its genetic influence, but only half of its shared environmental influence, with religiousness.

  2. Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones.

    PubMed

    Wolterink-van Loo, Suzanne; Escamilla Ayala, Abril A; Hooykaas, Paul J J; van Heusden, G Paul H

    2015-02-01

    Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA.

  3. Agrobacterium persistence in plant tissues after transformation.

    PubMed

    Cubero, Jaime; López, María M

    2005-01-01

    Agrobacterium spp. are routinely used in plant transformation to introduce genes of interest in valuable economic species. However, several agrobacteria species are also plant pathogens with ability to survive in different environments including the inner part of the plants. To avoid the release of genetic modified bacteria a successful plant transformation protocol must include the total elimination of agrobacteria by the use of antibiotics. Because sometimes these antibiotics failed in removing the bacteria entirely, confirmation of agrobacteria absence after plant transformation and regeneration is required. Different methodologies can be used for this purpose: isolation techniques followed by identification are used if detection of viable and culturable bacteria is necessary and techniques based on the polymerase chain reaction can be used to detect agrobacteria independently of their physiological state. Here we present several protocols to detect Agrobacterium in tissues of transformed plants as well as methods to identify the strains isolated. These identification methods can help to elucidate if they are the engineered bacteria used in the transformation process or just part of the natural endophytic microbiota.

  4. Nonsense-mediated decay in genetic disease: friend or foe?

    PubMed

    Miller, Jake N; Pearce, David A

    2014-01-01

    Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, there is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target.

  5. Nonsense-Mediated Decay in Genetic Disease: Friend or Foe?

    PubMed Central

    Miller, Jake N.; Pearce, David A.

    2014-01-01

    Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, there is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target. PMID:25485595

  6. Genetic immunization with plasmid DNA mediated by electrotransfer.

    PubMed

    Rochard, Alice; Scherman, Daniel; Bigey, Pascal

    2011-07-01

    The concept of DNA immunization was first advanced in the early 1990s, but was not developed because of an initial lack of efficiency. Recent technical advances in plasmid design and gene delivery techniques have allowed renewed interest in the idea. Particularly, a better understanding of genetic immunization has led to construction of optimized plasmids and the use of efficient molecular adjuvants. The field also took great advantage of new delivery techniques such as electrotransfer. This is a simple physical technique consisting of injecting plasmid DNA into a target tissue and applying an electric field, allowing up to a thousandfold more expression of the transgene than naked DNA. DNA immunization mediated by electrotransfer is now effective in a variety of preclinical models against infectious or acquired diseases such as cancer or autoimmune diseases, and is making its way through the clinics in several ongoing phase I human clinical trials. This review will briefly describe genetic immunization mediated by electrotransfer and the main fields of application.

  7. When plant virology met Agrobacterium: the rise of the deconstructed clones.

    PubMed

    Peyret, Hadrien; Lomonossoff, George P

    2015-10-01

    In the early days of molecular farming, Agrobacterium-mediated stable genetic transformation and the use of plant virus-based vectors were considered separate and competing technologies with complementary strengths and weaknesses. The demonstration that 'agroinfection' was the most efficient way of delivering virus-based vectors to their target plants blurred the distinction between the two technologies and permitted the development of 'deconstructed' vectors based on a number of plant viruses. The tobamoviruses, potexviruses, tobraviruses, geminiviruses and comoviruses have all been shown to be particularly well suited to the development of such vectors in dicotyledonous plants, while the development of equivalent vectors for use in monocotyledonous plants has lagged behind. Deconstructed viral vectors have proved extremely effective at the rapid, high-level production of a number of pharmaceutical proteins, some of which are currently undergoing clinical evaluation.

  8. An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.

    PubMed

    Ma, Lay-Sun; Lin, Jer-Sheng; Lai, Erh-Min

    2009-07-01

    An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpL(M), an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL(M) and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of beta-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL(M) is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL(M) mutants with substitutions or deletions in the Walker A motif failed to complement the impL(M) deletion mutant for Hcp secretion, which provided evidence that ImpL(M) may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL(M) and another essential T6SS component, ImpK(L). Topology and biochemical fractionation analyses suggested that ImpK(L) is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL(M)-ImpK(L) interaction domains suggested that ImpL(M) interacts with ImpK(L) via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL(M) interacts with ImpK(L), and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.

  9. GLYCINE RESISTANCE IN AGROBACTERIUM TUMEFACIENS

    PubMed Central

    Beardsley, Robert E.

    1962-01-01

    Beardsley, Robert E. (Manhattan College, New York, N. Y.). Glycine resistance in Agrobacterium tumefaciens. J. Bacteriol. 83:6–13. 1962.—The application of the fluctuation test of Luria and Delbrück to the distribution of glycine-resistant bacteria among cultures of Agrobacterium tumefaciens strain B6 indicates that resistance arises by mutation in the absence of glycine. On glycine-supplemented medium, additional resistant colonies arise during prolonged periods of incubation. Their appearance is proceded by L-form growth. In general, the number of generations over which glycine resistance is inherited in the absence of glycine is increased by serial transfers on the selection medium. In liquid medium containing glycine, sensitive bacteria form spheroplasts. Resistant bacteria continue to grow as rod forms. In the medium employed, spheroplasts are unstable. Images PMID:13866159

  10. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

    PubMed Central

    Srinivasan, Ramachandran

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer. PMID:27351975

  11. Plant responses to Agrobacterium tumefaciens and crown gall development

    PubMed Central

    Gohlke, Jochen; Deeken, Rosalia

    2014-01-01

    Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (“omic”) approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant. PMID:24795740

  12. Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens.

    PubMed

    Martínez-Cruz, Jesús; Romero, Diego; de Vicente, Antonio; Pérez-García, Alejandro

    2017-03-01

    The obligate biotrophic fungal pathogen Podosphaera xanthii is the main causal agent of powdery mildew in cucurbit crops all over the world. A major limitation of molecular studies of powdery mildew fungi (Erysiphales) is their genetic intractability. In this work, we describe a robust method based on the promiscuous transformation ability of Agrobacterium tumefaciens for reliable transformation of P. xanthii. The A. tumefaciens-mediated transformation (ATMT) system yielded transformants of P. xanthii with diverse transferred DNA (T-DNA) constructs. Analysis of the resultant transformants showed the random integration of T-DNA into the P. xanthii genome. The integrations were maintained in successive generations in the presence of selection pressure. Transformation was found to be transient, because in the absence of selection agent, the introduced genetic markers were lost due to excision of T-DNA from the genome. The ATMT system represents a potent tool for genetic manipulation of P. xanthii and will likely be useful for studying other biotrophic fungi. We hope that this method will contribute to the development of detailed molecular studies of the intimate interaction established between powdery mildew fungi and their host plants.

  13. Overexpression of several Arabidopsis histone genes increases Agrobacterium-medicated transformation and transgene expression in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis histone H2A-1 is important for Agrobacterium-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, in the rat5 mutant results in decreased T-(transferred) DNA integration into the plant genome, whereas over-expression of HTA1 increases transformation freq...

  14. Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In cer...

  15. Are Associations between Parental Divorce and Children's Adjustment Genetically Mediated? An Adoption Study.

    ERIC Educational Resources Information Center

    O'Connor, Thomas G.; Caspi, Avshalom; DeFries, John C.; Plomin, Robert

    2000-01-01

    Data from Colorado Adoption Project were used to examine hypothesis that association between parental divorce and children's adjustment is mediated by genetic factors. Findings for psychopathology were consistent with an environmentally mediated explanation for the association. Findings for achievement and social adjustment were consistent with a…

  16. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  17. Translating Genetic Research into Preventive Intervention: The Baseline Target Moderated Mediator Design.

    PubMed

    Howe, George W; Beach, Steven R H; Brody, Gene H; Wyman, Peter A

    2015-01-01

    In this paper we present and discuss a novel research approach, the baseline target moderated mediation (BTMM) design, that holds substantial promise for advancing our understanding of how genetic research can inform prevention research. We first discuss how genetically informed research on developmental psychopathology can be used to identify potential intervention targets. We then describe the BTMM design, which employs moderated mediation within a longitudinal study to test whether baseline levels of intervention targets moderate the impact of the intervention on change in that target, and whether change in those targets mediates causal impact of preventive or treatment interventions on distal health outcomes. We next discuss how genetically informed BTMM designs can be applied to both microtrials and full-scale prevention trials. We use simulated data to illustrate a BTMM, and end with a discussion of some of the advantages and limitations of this approach.

  18. Translating Genetic Research into Preventive Intervention: The Baseline Target Moderated Mediator Design

    PubMed Central

    Howe, George W.; Beach, Steven R. H.; Brody, Gene H.; Wyman, Peter A.

    2016-01-01

    In this paper we present and discuss a novel research approach, the baseline target moderated mediation (BTMM) design, that holds substantial promise for advancing our understanding of how genetic research can inform prevention research. We first discuss how genetically informed research on developmental psychopathology can be used to identify potential intervention targets. We then describe the BTMM design, which employs moderated mediation within a longitudinal study to test whether baseline levels of intervention targets moderate the impact of the intervention on change in that target, and whether change in those targets mediates causal impact of preventive or treatment interventions on distal health outcomes. We next discuss how genetically informed BTMM designs can be applied to both microtrials and full-scale prevention trials. We use simulated data to illustrate a BTMM, and end with a discussion of some of the advantages and limitations of this approach. PMID:26779062

  19. Utilization of Octopine and Nopaline by Agrobacterium

    PubMed Central

    Lippincott, James A.; Beiderbeck, Rolf; Lippincott, Barbara B.

    1973-01-01

    Tests for utilization of d-octopine and nopaline in defined media containing a carbon and nitrogen source were made on 60 strains of Agrobacterium representing four species and on a representative of each of five species of Rhizobium. Among 46 virulent strains of Agrobacterium, only two strains were found which utilized neither compound, while three strains were found which could utilize both. Of the remaining virulent strains, 27 utilized octopine and 14 utilized nopaline. Each of six strains of A. rhizogenes tested utilized only octopine but at a slower rate relative to growth than most A. tumefaciens. All eight of the A. radiobacter strains failed to utilize either compound, as did four of six nonvirulent strains of A. tumefaciens. The rhizobia did not utilize octopine or, with the possible exception of R. japonicum, nopaline. Virulence in the genus Agrobacterium is concluded to be highly correlated with the ability to utilize one or both of these compounds. PMID:4745420

  20. Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection.

    PubMed

    RamanaRao, Mangu Venkata; Veluthambi, Karuppannan

    2010-05-01

    Transient selection involving the bar gene and non-conditional negative selection against stable T-DNA integration through the use of the Mungbean yellow mosaic virus (MYMV) transcriptional activator protein gene (TrAP) were used in a novel co-transformation strategy to generate selectable marker gene (SMG)-eliminated transgenic tobacco plants in the T(0) generation itself. Two compatible binary plasmids, pCam-bar-TrAP-gus harbouring bar as an SMG and the MYMV TrAP gene as a non-conditional negative selectable marker, and pGA472 with the nptII gene as an unselected experimental gene of interest (GOI) were placed in the Agrobacterium tumefaciens strain EHA105 and used for co-transformation. Transient selection with 5 mg l(-1) phosphinothricin (PPT) for 2-4 weeks and subsequent establishment in a PPT-minus medium yielded 114 plants from 200 leaf discs. The unselected nptII gene was detected by Southern blot analysis in 13 plants, revealing a co-transformation efficiency of 11.5%. Five of these plants harboured only the nptII gene (GOI) and not the bar gene (SMG). Thus, SMG elimination was achieved in the T(0) generation itself in 4.4% (5/114) of plants, which were transiently selected for 2-4 weeks on PPT. MYMV TrAP, a non-conditional negative selectable marker, effectively reduced the recovery of plants with stable integration of the SMG (bar).

  1. Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

    PubMed Central

    Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

    2012-01-01

    The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

  2. Personality Mediation of Genetic Effects on Attention-Deficit/Hyperactivity Disorder

    ERIC Educational Resources Information Center

    Martel, Michelle M.; Nikolas, Molly; Jernigan, Katherine; Friderici, Karen; Nigg, Joel T.

    2010-01-01

    Personality traits may be viable candidates for mediators of the relationship between genetic risk and ADHD. Participants were 578 children (331 boys; 320 children with ADHD) between the ages of six and 18. Parents and teachers completed a comprehensive, multi-stage diagnostic procedure to assess ADHD and comorbid disorders. Mother completed the…

  3. GENETIC INFLUENCES ON IN VTIRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE

    EPA Science Inventory

    GENETIC INFLUENCES ON IN VITRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE.
    JA Dye, JH Richards, DA Andrews, UP Kodavanti. US EPA, RTP, NC, USA.

    Particulate matter (PM) air pollution is capable of damaging the airway epitheli...

  4. Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

    PubMed Central

    Kuraguchi, Mari; Ohene-Baah, Nana Yaw; Sonkin, Dmitriy; Bronson, Roderick Terry; Kucherlapati, Raju

    2009-01-01

    Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; ApcCKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC. PMID:19197353

  5. Viral Vector-Mediated Antisense Therapy for Genetic Diseases

    PubMed Central

    Imbert, Marine; Dias-Florencio, Gabriella; Goyenvalle, Aurélie

    2017-01-01

    RNA plays complex roles in normal health and disease and is becoming an important target for therapeutic intervention; accordingly, therapeutic strategies that modulate RNA function have gained great interest over the past decade. Antisense oligonucleotides (AOs) are perhaps the most promising strategy to modulate RNA expression through a variety of post binding events such as gene silencing through degradative or non-degradative mechanisms, or splicing modulation which has recently demonstrated promising results. However, AO technology still faces issues like poor cellular-uptake, low efficacy in target tissues and relatively rapid clearance from the circulation which means repeated injections are essential to complete therapeutic efficacy. To overcome these limitations, viral vectors encoding small nuclear RNAs have been engineered to shuttle antisense sequences into cells, allowing appropriate subcellular localization with pre-mRNAs and permanent correction. In this review, we outline the different strategies for antisense therapy mediated by viral vectors and provide examples of each approach. We also address the advantages and limitations of viral vector use, with an emphasis on their clinical application. PMID:28134780

  6. Metabolic factors and genetic risk mediate familial type 2 diabetes risk in the Framingham Heart Study

    PubMed Central

    Raghavan, Sridharan; Porneala, Bianca; McKeown, Nicola; Fox, Caroline S.; Dupuis, Josée; Meigs, James B.

    2015-01-01

    Aims/hypothesis Type 2 diabetes mellitus in parents is a strong determinant of diabetes risk in their offspring. We hypothesise that offspring diabetes risk associated with parental diabetes is mediated by metabolic risk factors. Methods We studied initially non-diabetic participants of the Framingham Offspring Study. Metabolic risk was estimated using beta cell corrected insulin response (CIR), HOMA-IR or a count of metabolic syndrome components (metabolic syndrome score [MSS]). Dietary risk and physical activity were estimated using questionnaire responses. Genetic risk score (GRS) was estimated as the count of 62 type 2 diabetes risk alleles. The outcome of incident diabetes in offspring was examined across levels of parental diabetes exposure, accounting for sibling correlation and adjusting for age, sex and putative mediators. The proportion mediated was estimated by comparing regression coefficients for parental diabetes with (βadj) and without (βunadj) adjustments for CIR, HOMA-IR, MSS and GRS (percentage mediated = 1 – βadj / βunadj). Results Metabolic factors mediated 11% of offspring diabetes risk associated with parental diabetes, corresponding to a reduction in OR per diabetic parent from 2.13 to 1.96. GRS mediated 9% of risk, corresponding to a reduction in OR per diabetic parent from 2.13 to 1.99. Conclusions/interpretation Metabolic risk factors partially mediated offspring type 2 diabetes risk conferred by parental diabetes to a similar magnitude as genetic risk. However, a substantial proportion of offspring diabetes risk associated with parental diabetes remains unexplained by metabolic factors, genetic risk, diet and physical activity, suggesting that important familial influences on diabetes risk remain undiscovered. PMID:25619168

  7. Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agrobacterium rhizogenes-mediated induction of transgenic hairy roots was previously demonstrated in Vitis vinifera L. and a few other Vitis species. In this study, 13 Vitis species, including V. aestivalis, V. afghanistan, V. champinii, V. doaniana, V. flexuosa, V. labrusca, V. nesbittiana, V. pal...

  8. Genetic Background, Maternal Age, and Interaction Effects Mediate Rates of Crossing Over in Drosophila melanogaster Females.

    PubMed

    Hunter, Chad M; Robinson, Matthew C; Aylor, David L; Singh, Nadia D

    2016-05-03

    Meiotic recombination is a genetic process that is critical for proper chromosome segregation in many organisms. Despite being fundamental for organismal fitness, rates of crossing over vary greatly between taxa. Both genetic and environmental factors contribute to phenotypic variation in crossover frequency, as do genotype-environment interactions. Here, we test the hypothesis that maternal age influences rates of crossing over in a genotypic-specific manner. Using classical genetic techniques, we estimated rates of crossing over for individual Drosophila melanogaster females from five strains over their lifetime from a single mating event. We find that both age and genetic background significantly contribute to observed variation in recombination frequency, as do genotype-age interactions. We further find differences in the effect of age on recombination frequency in the two genomic regions surveyed. Our results highlight the complexity of recombination rate variation and reveal a new role of genotype by maternal age interactions in mediating recombination rate.

  9. Agrobacterium tumefaciens responses to plant-derived signaling molecules.

    PubMed

    Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

    2014-01-01

    As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including γ-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium-plant interactions.

  10. Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains.

    PubMed

    Mhamdi, Ridha; Mrabet, Moncef; Laguerre, Gisèle; Tiwari, Ravi; Aouani, Mohamed Elarbi

    2005-02-01

    Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.

  11. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    PubMed

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  12. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity.

    PubMed

    Lippincott, B B; Lippincott, J A

    1966-10-01

    Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

  13. Two marine Agrobacterium producers of sesbanimide antibiotics.

    PubMed

    Acebal, C; Alcazar, R; Cañedo, L M; de la Calle, F; Rodriguez, P; Romero, F; Fernandez Puentes, J L

    1998-01-01

    Sesbanimides are cytotoxic compounds, originally isolated in 1983 from seeds of the leguminous plants Sesbania drummondii and Sesbania punicea. In this paper we describe the bacterial production of sesbanimides by two "marine Agrobacterium"; strain PH-103 which produces Sesbanimide-A and strain PH-A034C which produces Sesbanimide-C. The isolation and taxonomy of the producing microorganisms, fermentation and isolation of sesbanimides are reported.

  14. Genetic and biochemical dissection of transgenic RNA-mediated virus resistance.

    PubMed Central

    Goodwin, J; Chapman, K; Swaney, S; Parks, T D; Wernsman, E A; Dougherty, W G

    1996-01-01

    RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines. PMID:8597662

  15. Genetic and environmental mediation between measures of personality and family environment in twins reared together.

    PubMed

    Kandler, Christian; Riemann, Rainer; Kämpfe, Nicole

    2009-01-01

    In this study we analyzed the etiology of the relationship between personality traits and retrospectively recalled family environment. The data of 226 identical and 168 fraternal twin pairs reared together from the Jena twin study of social attitudes were available. Personality traits were measured using the self- and peer report versions of the German NEO-personality inventory-revised. A German version of Blocks Environmental Questionnaire was applied to measure two broad dimensions of the family environment retrospectively: support and organization. We could replicate earlier findings that retrospective reports of these family environment dimensions were in part genetically influenced. A total of 66% of the genetic variance in support and 24% in organization could be accounted for by heritable variance in self-rated personality. That was replicated by using peer reports of personality, 41% explained genetic variance in support and 17% in organization. Environmental mediations were negligible. This indicates that the relationship between personality and retrospectively recalled family environment is largely genetically mediated.

  16. The Association Between Conduct Problems and Maltreatment: Testing Genetic and Environmental Mediation

    PubMed Central

    Rhee, Soo Hyun; Silvern, Louise E.; Haberstick, Brett C.; Hopfer, Christian; Lessem, Jeffrey M.; Hewitt, John K.

    2011-01-01

    It is often assumed that childhood maltreatment causes conduct problems via an environmentally mediated process. However, the association may be due alternatively to either a nonpassive gene-environment correlation, in which parents react to children’s genetically-influenced conduct problems by maltreating them, or a passive gene-environment correlation, in which parents’ tendency to engage in maltreatment and children’s conduct problems are both influenced by a hereditary vulnerability to antisocial behavior (i.e. genetic mediation). The present study estimated the contribution of these processes to the association between maltreatment and conduct problems. Bivariate behavior genetic analyses were conducted on approximately 1,650 twin and sibling pairs drawn from a large longitudinal study of adolescent health (Add Health). The correlation between maltreatment and conduct problems was small; much of the association between maltreatment and conduct problems was due to a nonpassive gene-environment correlation. Results were more consistent with the hypothesis that parents respond to children’s genetically-influenced conduct problems by maltreating them than the hypothesis that maltreatment causes conduct problems. PMID:20024671

  17. Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose.

    PubMed

    Shin, Hyun-Dong; Liu, Long; Kim, Mi-Kyoung; Park, Yong-Il; Chen, Rachel

    2016-09-01

    Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

  18. Genetic diversity predicts pathogen resistance and cell-mediated immunocompetence in house finches

    PubMed Central

    Hawley, Dana M; Sydenstricker, Keila V; Kollias, George V; Dhondt, André A

    2005-01-01

    Evidence is accumulating that genetic variation within individual hosts can influence their susceptibility to pathogens. However, there have been few opportunities to experimentally test this relationship, particularly within outbred populations of non-domestic vertebrates. We performed a standardized pathogen challenge in house finches (Carpodacus mexicanus) to test whether multilocus heterozygosity across 12 microsatellite loci predicts resistance to a recently emerged strain of the bacterial pathogen, Mycoplasma gallisepticum (MG). We simultaneously tested whether the relationship between heterozygosity and pathogen susceptibility is mediated by differences in cell-mediated or humoral immunocompetence. We inoculated 40 house finches with MG under identical conditions and assayed both humoral and cell-mediated components of the immune response. Heterozygous house finches developed less severe disease when infected with MG, and they mounted stronger cell-mediated immune responses to phytohaemagglutinin. Differences in cell-mediated immunocompetence may, therefore, partly explain why more heterozygous house finches show greater resistance to MG. Overall, our results underscore the importance of multilocus heterozygosity for individual pathogen resistance and immunity. PMID:17148199

  19. Host Genetic Control of the Microbiota Mediates the Drosophila Nutritional Phenotype

    PubMed Central

    Chaston, John M.; Dobson, Adam J.; Newell, Peter D.

    2015-01-01

    A wealth of studies has demonstrated that resident microorganisms (microbiota) influence the pattern of nutrient allocation to animal protein and energy stores, but it is unclear how the effects of the microbiota interact with other determinants of animal nutrition, including animal genetic factors and diet. Here, we demonstrate that members of the gut microbiota in Drosophila melanogaster mediate the effect of certain animal genetic determinants on an important nutritional trait, triglyceride (lipid) content. Parallel analysis of the taxonomic composition of the associated bacterial community and host nutritional indices (glucose, glycogen, triglyceride, and protein contents) in multiple Drosophila genotypes revealed significant associations between the abundance of certain microbial taxa, especially Acetobacteraceae and Xanthamonadaceae, and host nutritional phenotype. By a genome-wide association study of Drosophila lines colonized with a defined microbiota, multiple host genes were statistically associated with the abundance of one bacterium, Acetobacter tropicalis. Experiments using mutant Drosophila validated the genetic association evidence and reveal that host genetic control of microbiota abundance affects the nutritional status of the flies. These data indicate that the abundance of the resident microbiota is influenced by host genotype, with consequent effects on nutrient allocation patterns, demonstrating that host genetic control of the microbiome contributes to the genotype-phenotype relationship of the animal host. PMID:26567306

  20. Communicating about the risks and benefits of genetically modified foods: the mediating role of trust.

    PubMed

    Frewer, Lynn J; Scholderer, Joachim; Bredahl, Lone

    2003-12-01

    Recent research suggests that public attitudes toward emerging technologies are mainly driven by trust in the institutions promoting and regulating these technologies. Alternative views maintain that trust should be seen as a consequence rather than a cause of such attitudes. To test its actual role, direct as well as mediating effects of trust were tested in an attitude change experiment involving 1,405 consumers from Denmark, Germany, Italy, and the United Kingdom. After prior attitudes to genetic modification in food production had been assessed, participants received different information materials (either product-specific information or balanced/general information about genetic modification in food production) and were asked to evaluate different types of genetically modified foods (either beer or yoghurt). The information materials were attributed to different information sources (either an industry association, a consumer organization, or a government source). After completion, perceived risk and perceived benefit were assessed, and participants indicated their trust in the information sources to which the materials had been attributed. Direct and trust-mediated attitude change effects were estimated in a multi-sample structural equation model. The results showed that information provision had little effect on people's attitudes toward genetically modified foods, and that perceptions of information source characteristics contributed very little to attitude change. Furthermore, the type of information strategy adopted had almost no impact on postexperimental attitudes. The extent to which people trusted the information sources appeared to be driven by people's attitudes to genetically modified foods, rather than trust influencing the way that people reacted to the information. Trust was not driving risk perception-rather, attitudes were informing perceptions of the motivation of the source providing the information.

  1. Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa).

    PubMed

    Xu, Meimei; Galhano, Rita; Wiemann, Philipp; Bueno, Emilio; Tiernan, Mollie; Wu, William; Chung, Ill-Min; Gershenzon, Jonathan; Tudzynski, Bettina; Sesma, Ane; Peters, Reuben J

    2012-02-01

    • There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). • However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. • The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). • Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.

  2. Genetic contribution to brachial artery flow-mediated dilation: The Northern Manhattan Family Study

    PubMed Central

    Suzuki, Keiko; Juo, Suh-Hang Hank; Rundek, Tanja; Boden-Albala, Bernadette; Disla, Norbelina; Liu, Rui; Park, Naeun; Di Tullio, Marco R.; Sacco, Ralph L.; Homma, Shunichi

    2007-01-01

    Background Brachial artery flow-mediated dilation (FMD) is a non-invasive measure of endothelial function. Endothelial dysfunction has been associated with traditional vascular risk factors and increased risk of cardiovascular disease. The importance of genetic contribution to FMD and baseline brachial artery diameter has not been shown in Hispanic populations. The purpose of this study was to estimate the heritability of FMD. Methods Flow mediated dilation and brachial artery diameter were measured in a subset of Caribbean Hispanic families from the ongoing Northern Manhattan Family Study (NOMAFS), which studies the contribution of genetics to stroke and cardiovascular risk factors. The age- and sex-adjusted heritability of FMD was estimated using variance component methods. Results The current data include 620 subjects (97 probands and 523 relatives) from 97 families. The age and sex-adjusted heritability of brachial artery diameter was 0.57 (p < 0.01). The age- and sex-adjusted heritability of FMD was 0.20 (p = 0.01). After additional adjustment for systolic and diastolic blood pressure, body mass index, smoking, lipid, diabetes mellitus, medication, and baseline brachial artery diameter, the heritability of FMD was 0.17 (p = 0.01). Conclusions We found modest heritability of FMD. FMD might be a reasonable phenotype for further investigation of genetic contribution to atherosclerosis. PMID:17462653

  3. Genetic variation in MAOA modulates ventromedial prefrontal circuitry mediating individual differences in human personality.

    PubMed

    Buckholtz, J W; Callicott, J H; Kolachana, B; Hariri, A R; Goldberg, T E; Genderson, M; Egan, M F; Mattay, V S; Weinberger, D R; Meyer-Lindenberg, A

    2008-03-01

    Little is known about neural mechanisms underlying human personality and temperament, despite their considerable importance as highly heritable risk mediators for somatic and psychiatric disorders. To identify these circuits, we used a combined genetic and imaging approach focused on Monoamine Oxidase A (MAOA), encoding a key enzyme for monoamine metabolism previously associated with temperament and antisocial behavior. Male carriers of a low-expressing genetic variant exhibited dysregulated amygdala activation and increased functional coupling with ventromedial prefrontal cortex (vmPFC). Stronger coupling predicted increased harm avoidance and decreased reward dependence scores, suggesting that this circuitry mediates a part of the association of MAOA with these traits. We utilized path analysis to parse the effective connectivity within this system, and provide evidence that vmPFC regulates amygdala indirectly by influencing rostral cingulate cortex function. Our data implicate a neural circuit for variation in human personality under genetic control, provide an anatomically consistent mechanism for vmPFC-amygdala interactions underlying this variation, and suggest a role for vmPFC as a superordinate regulatory area for emotional arousal and social behavior.

  4. Mechanisms and regulation of surface interactions and biofilm formation in Agrobacterium

    PubMed Central

    Heindl, Jason E.; Wang, Yi; Heckel, Brynn C.; Mohari, Bitan; Feirer, Nathan; Fuqua, Clay

    2014-01-01

    For many pathogenic bacteria surface attachment is a required first step during host interactions. Attachment can proceed to invasion of host tissue or cells or to establishment of a multicellular bacterial community known as a biofilm. The transition from a unicellular, often motile, state to a sessile, multicellular, biofilm-associated state is one of the most important developmental decisions for bacteria. Agrobacterium tumefaciens genetically transforms plant cells by transfer and integration of a segment of plasmid-encoded transferred DNA (T-DNA) into the host genome, and has also been a valuable tool for plant geneticists. A. tumefaciens attaches to and forms a complex biofilm on a variety of biotic and abiotic substrates in vitro. Although rarely studied in situ, it is hypothesized that the biofilm state plays an important functional role in the ecology of this organism. Surface attachment, motility, and cell division are coordinated through a complex regulatory network that imparts an unexpected asymmetry to the A. tumefaciens life cycle. In this review, we describe the mechanisms by which A. tumefaciens associates with surfaces, and regulation of this process. We focus on the transition between flagellar-based motility and surface attachment, and on the composition, production, and secretion of multiple extracellular components that contribute to the biofilm matrix. Biofilm formation by A. tumefaciens is linked with virulence both mechanistically and through shared regulatory molecules. We detail our current understanding of these and other regulatory schemes, as well as the internal and external (environmental) cues mediating development of the biofilm state, including the second messenger cyclic-di-GMP, nutrient levels, and the role of the plant host in influencing attachment and biofilm formation. A. tumefaciens is an important model system contributing to our understanding of developmental transitions, bacterial cell biology, and biofilm formation

  5. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    SciTech Connect

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; Vogel, John P.; Thilmony, Roger

    2016-05-24

    In this study, the genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

  6. Why did eukaryotes evolve only once? Genetic and energetic aspects of conflict and conflict mediation.

    PubMed

    Blackstone, Neil W

    2013-07-19

    According to multi-level theory, evolutionary transitions require mediating conflicts between lower-level units in favour of the higher-level unit. By this view, the origin of eukaryotes and the origin of multicellularity would seem largely equivalent. Yet, eukaryotes evolved only once in the history of life, whereas multicellular eukaryotes have evolved many times. Examining conflicts between evolutionary units and mechanisms that mediate these conflicts can illuminate these differences. Energy-converting endosymbionts that allow eukaryotes to transcend surface-to-volume constraints also can allocate energy into their own selfish replication. This principal conflict in the origin of eukaryotes can be mediated by genetic or energetic mechanisms. Genome transfer diminishes the heritable variation of the symbiont, but requires the de novo evolution of the protein-import apparatus and was opposed by selection for selfish symbionts. By contrast, metabolic signalling is a shared primitive feature of all cells. Redox state of the cytosol is an emergent feature that cannot be subverted by an individual symbiont. Hypothetical scenarios illustrate how metabolic regulation may have mediated the conflicts inherent at different stages in the origin of eukaryotes. Aspects of metabolic regulation may have subsequently been coopted from within-cell to between-cell pathways, allowing multicellularity to emerge repeatedly.

  7. Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

    PubMed

    Xu, Junyi; Zheng, Qiuyue; Yu, Ling; Liu, Ran; Zhao, Xin; Wang, Gang; Wang, Qinghua; Cao, Jijuan

    2013-11-01

    The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg(-1) GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

  8. Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA).

    PubMed

    Zanetta, Chiara; Riboldi, Giulietta; Nizzardo, Monica; Simone, Chiara; Faravelli, Irene; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-02-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell-mediated approaches for the treatment of SMA.

  9. Studies on nonsense mediated decay reveal novel therapeutic options for genetic diseases.

    PubMed

    Bashyam, Murali D

    2009-01-01

    Scientific breakthroughs have often led to commercially viable patents mainly in the field of engineering. Commercialization in the field of medicine has been restricted mostly to machinery and engineering on the one hand and therapeutic drugs for common chronic ailments such as cough, cold, headache, etc, on the other. Sequencing of the human genome has attracted the attention of pharmaceutical companies and now biotechnology has become a goldmine for commercialization of products and processes. Recent advances in our understanding of basic biological processes have resulted in the opening of new avenues for treatment of human genetic diseases, especially single gene disorders. A significant proportion of human genetic disorders have been shown to be caused due to degradation of transcripts for specific genes through a process called nonsense mediated decay (NMD). The modulation of NMD provides a viable therapeutic option for treatment of several genetic disorders and therefore has been a good prospect for patenting and commercialization. In this review the molecular basis for NMD and attempts to treat genetic diseases which result from NMD are discussed.

  10. Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus.

    PubMed

    Park, Byong-Jin; Liu, Zaochang; Kanno, Akira; Kameya, Toshiaki

    2005-10-01

    A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T(1) plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.

  11. Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

    PubMed

    Hwang, Hau-Hsuan; Liu, Yin-Tzu; Huang, Si-Chi; Tung, Chin-Yi; Huang, Fan-Chen; Tsai, Yun-Long; Cheng, Tun-Fang; Lai, Erh-Min

    2015-02-01

    Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

  12. Genetic Transformation of Switchgrass

    NASA Astrophysics Data System (ADS)

    Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

    Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

  13. Testing for the mediating role of endophenotypes using molecular genetic data in a twin study of ADHD traits

    PubMed Central

    Pinto, Rebecca; Asherson, Philip; Ilott, Nicholas; Cheung, Celeste H. M.

    2016-01-01

    Family and twin studies have identified endophenotypes that capture familial and genetic risk in attention‐deficit/hyperactivity disorder (ADHD), but it remains unclear if they lie on the causal pathway. Here, we illustrate a stepwise approach to identifying intermediate phenotypes. First, we use previous quantitative genetic findings to delineate the expected pattern of genetically correlated phenotypes. Second, we identify overlapping genetic associations with ADHD‐related quantitative traits. Finally, we test for the mediating role of associated endophenotypes. We applied this approach to a sample of 1,312 twins aged 7–10. Based on previous twin model‐fitting analyses, we selected hyperactivity–impulsivity, inattention, reading difficulties (RD), reaction time variability (RTV) and commission errors (CE), and tested for association with selected ADHD risk alleles. For nominally significant associations with both a symptom and a cognitive variable, matching the expected pattern based on previous genetic correlations, we performed mediation analysis to distinguish pleiotropic from mediating effects. The strongest association was observed for the rs7984966 SNP in the serotonin receptor gene (HTR2A), and RTV (P = 0.007; unadjusted for multiple testing). Mediation analysis suggested that CE (38%) and RTV (44%) substantially mediated the association between inattention and the T‐allele of SNP rs3785157 in the norepinephrine transporter gene (SLC6A2) and the T‐allele of SNP rs7984966 in HTR2A, respectively. The SNPs tag risk‐haplotypes but are not thought to be functionally significant. While these exploratory findings are preliminary, requiring replication, this study demonstrates the value of this approach that can be adapted to the investigation of multiple genetic markers and polygenic risk scores. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc. PMID:27230021

  14. Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients.

    PubMed

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-06-01

    Previous clinical trials based on the genetic correction of purified CD34(+) cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

  15. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients.

    PubMed

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Lozano, M Luz; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-06-01

    Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

  16. Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

    PubMed Central

    Jacome, Ariana; Navarro, Susana; Río, Paula; Yañez, Rosa M; González-Murillo, Africa; Luz Lozano, M; Lamana, Maria Luisa; Sevilla, Julian; Olive, Teresa; Diaz-Heredia, Cristina; Badell, Isabel; Estella, Jesus; Madero, Luis; Guenechea, Guillermo; Casado, José; Segovia, Jose C; Bueren, Juan A

    2009-01-01

    Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34− mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs). PMID:19277017

  17. Fast recovery of transgenic submergence tolerant rice cultivars of North-East India by early co-cultivation of Agrobacterium with pre-cultured callus.

    PubMed

    Nguyen, Van Cuu; Nguyen, Van Khiem; Singh, Chongtham Henary; Devi, Guruaribam Shantibala; Reddy, Vanga Siva; Leelavathi, Sadhu

    2017-01-01

    Agro-climatic conditions of North-East India are very complex and rice cultivars present in the region have been adapted to grow under harsh environmental conditions. Germplasm present in the region is considered to possess several important and unique traits that are of importance in rice improvement programs. Genetic engineering is a powerful tool to introduce new traits into crop plants. However, not much information is available on the methods to introduce foreign genes into North-East rice cultivars. Therefore, the main objective of this study is to develop transformation procedures for fast recovery of transgenic plants from North-East rice cultivars. To achieve this objective, a systematic study was carried out to identify media components and culture conditions for efficient embryogenic callus induction from the mature seeds and differentiation of callus into plantlets from two North-East deep water rice cultivars, Taothabi and Khongan. Also, role of preculture of callus on Agrobacterium-mediated transformation was studied. Co-cultivation of Agrobacterium with 1-5 days precultured callus was found to result in high frequency of transformation. Detailed characterization of transgenic lines confirmed stable integration of transgenes and expression of reporter gfp gene. The whole process starting from callus induction to regenerating of transgenic rice plants that can be established in the soil was achieved in about 35-45 days. The procedures developed were found to be applicable to a popular variety IR 64. Therefore, methods developed in this study should be useful not only to introduce new traits quickly but also to validate the function(s) of several candidate gene(s) identified under the functional genomics of rice.

  18. [Obtaining transgenic rice plants and their progenies using Agrobacterium tumefaciens].

    PubMed

    Yin, Z C; Yang, F; Xu, Y; Li, B J

    1998-12-01

    Rice (Oriza sativa L.) suspension cells of Taipei 309 were co-cultivated with A. tumefaciens stran EHA101 harbouring binary vector pBYT2 for 3 days in the presence of vir inducer, 100 mumol/L acetosyringone (AS). After 2 months of continuous selection, 17 stable hygromycin-resistant, GUS-positive calli were recovered from 364 suspension cell clusters co-cultivated with A. tumefaciens. 10 putative transgenic R0 plants obtained from 8 tansformed calli and their progenies were analyzed for the integration and expression of foreign genes. Southern blot analysis of R0 and R1 generations indicated that foreign genes had been stably integrated in the genome of transgenic rice and sexually transmitted. One of the transgenic lines showed 5 copies of T-DNA integration, while the others had only one copy. Histochemical staining observation and fluorometric assay of GUS activity in transgenic rice cells and plants showed ubiquitin promoter from maize was highly effective in driving the expression of gus reporter gene in transgenic rice cells. GUS protein and its activity were also investigated through ndPAGE-X-Gluc staining assay, and it was found that the GUS protein in transgenic rice cells was smaller in size than the standard GUS protein (Sigma Co. G0786) but as large as that from E.coli HB101 (pBI121). This study suggested that Agrobacterium-mediated transformation of plant is an efficient and reliable method to introduce foreign genes into rice.

  19. A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology.

    PubMed

    Emami, Shahram; Yee, Muh-Ching; Dinneny, José R

    2013-01-01

    Tools that allow for rapid, accurate and inexpensive assembly of multi-component combinatorial libraries of DNA for transformation into plants will accelerate the progress of synthetic biology research. Recent innovations in molecular cloning methods has vastly expanded the repertoire with which plant biologists can engineer a transgene. Here we describe a new set of binary vectors for use in Agrobacterium-mediated plant transformation that utilizes the Golden-Gate Cloning approach. Our optimized protocol facilitates the rapid and inexpensive generation of multi-component transgenes for later introduction into plants.

  20. A Perspective on Hypericum perforatum Genetic Transformation.

    PubMed

    Hou, Weina; Shakya, Preeti; Franklin, Gregory

    2016-01-01

    Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering.

  1. A Perspective on Hypericum perforatum Genetic Transformation

    PubMed Central

    Hou, Weina; Shakya, Preeti; Franklin, Gregory

    2016-01-01

    Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering. PMID:27446112

  2. Genetically-mediated associations between measures of childhood character and academic achievement.

    PubMed

    Tucker-Drob, Elliot M; Briley, Daniel A; Engelhardt, Laura E; Mann, Frank D; Harden, K Paige

    2016-11-01

    Researchers and the general public have become increasingly intrigued by the roles that systematic tendencies toward thinking, feeling, and behaving might play in academic achievement. Some measures of constructs belonging to this group have been well studied in genetics and psychometrics, while much less is known about measures of other such constructs. The current study focuses on 7 character traits prominently featured in influential intervention-oriented and/or socialization theories of academic achievement: grit, intellectual curiosity, intellectual self-concept, mastery orientation, educational value, intelligence mindset, and test motivation. In a population-based sample of 811 school-aged twins and triplets from the Texas Twin Project, we tested (a) how each measure relates to indices of the Big Five personality traits, (b) how the measures relate to one another, (c) the extent to which each measure is associated with genetic and environmental influences and whether such influences operate through common dimensions of individual differences, and (d) the extent to which genetic and environmental factors mediate the relations between fluid intelligence, character measures, verbal knowledge, and academic achievement. We find moderate relations among the measures that can be captured by a highly heritable common dimension representing a mixture of Openness and Conscientiousness. Moreover, genetically influenced variance in the character measures is associated with multiple measures of verbal knowledge and academic achievement, even after controlling for fluid intelligence. In contrast, environmentally influenced variance in character is largely unrelated to knowledge and achievement outcomes. We propose that character measures popularly used in education may be best conceptualized as indexing facets of personality that are of particular relevance to academic achievement. (PsycINFO Database Record

  3. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens

    PubMed Central

    Czolkoss, Simon; Fritz, Christiane; Hölzl, Georg; Aktas, Meriyem

    2016-01-01

    Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants. PMID:27472399

  4. AMINO ACID CROSS RESISTANCE IN AGROBACTERIUM TUMEFACIENS

    PubMed Central

    Beardsley, Robert E.

    1962-01-01

    Beardsley, Robert E. (Manhattan College, New York, N. Y.). Amino acid cross resistance in Agrobacterium tumefaciens. J. Bacteriol. 84:1237–1240. 1962.—Resistant clones selected on medium supplemented with glycine were also resistant to d-methionine, d-valine, dl-norleucine, and dl-serine. Cross resistance was similarly exhibited by clones selected on d-methionine, d-valine, or dl-norleucine. Two types of resistant organisms were observed. One produced colonies containing normal rods on selection medium. The other produced translucent colonies containing L forms. Both grew as typical rods in unsupplemented medium. Some resistant clones did not produce a temperate phage carried by the parental strain, but these retained immunity to homologous phage. The toxicity of d-methionine and d-valine for nonresistant bacteria is not reversed by the l isomers. The lethal effects of toxic amino acids are additive. PMID:13969951

  5. Agrobacterium tumefaciens is a diazotrophic bacterium

    SciTech Connect

    Kanvinde, L.; Sastry, G.R.K. )

    1990-07-01

    This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.

  6. Plant viral vectors for delivery by Agrobacterium.

    PubMed

    Gleba, Yuri Y; Tusé, Daniel; Giritch, Anatoli

    2014-01-01

    Plant viral vectors delivered by Agrobacterium are the basis of several manufacturing processes that are currently in use for producing a wide range of proteins for multiple applications, including vaccine antigens, antibodies, protein nanoparticles such as virus-like particles (VLPs), and other protein and protein-RNA scaffolds. Viral vectors delivered by agrobacterial T-DNA transfer (magnifection) have also become important tools in research. In recent years, essential advances have been made both in the development of second-generation vectors designed using the 'deconstructed virus' approach, as well as in the development of upstream manufacturing processes that are robust and fully scalable. The strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA/DNA replicons; the bacteria are delivered into leaves by vacuum infiltration, and the viral machinery takes over from the point of T-DNA transfer to the plant cell nucleus, driving massive RNA and protein production and, if required, cell-to-cell spread of the replicons. Among the most often used viral backbones are those of the RNA viruses Tobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV), and the DNA geminivirus Bean yellow dwarf virus. Prototypes of industrial processes that provide for high yield, rapid scale up and fast manufacturing cycles have been designed, and several GMP-compliant and GMP-certified manufacturing facilities are in place. These efforts have been successful as evidenced by the fact that several antibodies and vaccine antigens produced by magnifection are currently in clinical development.

  7. Defining the Genetic Architecture Underlying Female- and Male-Mediated Nonrandom Mating and Seed Yield Traits in Arabidopsis1[OA

    PubMed Central

    Carlson, Ann Louise; Fitz Gerald, Jonathan Nesbit; Telligman, Megan; Roshanmanesh, Jacob; Swanson, Robert John

    2011-01-01

    Postpollination nonrandom mating among compatible mates is a widespread phenomenon in plants and is genetically undefined. In this study, we used the recombinant inbred line (RIL) population between Landsberg erecta and Columbia (Col) accessions of Arabidopsis (Arabidopsis thaliana) to define the genetic architecture underlying both female- and male-mediated nonrandom mating traits. To map the genetic loci responsible for male-mediated nonrandom mating, we performed mixed pollinations with Col and RIL pollen on Col pistils. To map the genetic loci responsible for female-mediated nonrandom mating, we performed mixed pollinations with Col and Landsberg erecta pollen on RIL pistils. With these data, we performed composite interval mapping to identify two quantitative trait loci (QTLs) that control male-mediated nonrandom mating. We detected epistatic interactions between these two loci. We also explored female- and male-mediated traits involved in seed yield in mixed pollinations. We detected three female QTLs and one male QTL involved in directing seed number per fruit. To our knowledge, the results of these experiments represent the first time the female and male components of seed yield and nonrandom mating have been separately mapped. PMID:22007025

  8. Genetic variation and population structure in the endangered Hermann's tortoise: the roles of geography and human-mediated processes.

    PubMed

    Perez, Melanie; Livoreil, Barbara; Mantovani, Sara; Boisselier, Marie-Catherine; Crestanello, Barbara; Abdelkrim, Jawad; Bonillo, Céline; Goutner, Vassilis; Lambourdière, Josie; Pierpaoli, Massimo; Sterijovski, Bogoljub; Tomovic, Ljiljana; Vilaça, Sibelle T; Mazzotti, Stefano; Bertorelle, Giorgio

    2014-01-01

    The Hermann's tortoise (Testudo hermanni) is an endangered land tortoise distributed in disjoint populations across Mediterranean Europe. We investigated its genetic variation by typing 1 mitochondrial locus and 9 nuclear microsatellites in approximately 300 individuals from 22 localities. Our goal was to understand the relative impact of natural and human-mediated processes in shaping the genetic structure and to identify the genetic priorities for the conservation of this species. We found that 1) all geographic areas are highly differentiated, mainly as a function of their distance but with a clear genetic discontinuity (F st values larger than 0.4) between the Eastern and the Western subspecies; 2) the contact zone between subspecies is located farthest to the west than previously believed, and it probably coincides with the delta of the largest Italian river; 3) extinction events due to climatic conditions in the Upper Palaeolithic and subsequent human-mediated translocations in the Neolithic possibly explain the unexpected similarity among Spain, Sicily, and Corsica. For conservation purposes, the large majority of genetic pools appears native although hybridization among subspecies, related to extensive 20th century trade of tortoises across Europe, is observed in Spain and some Italian samples. Most populations do not seem at immediate risk of low genetic variation, except the French population, which has very low nuclear genetic diversity (heterozygosity = 0.25) and where 50 out of 51 sampled animals shared the same mitochondrial sequence. In general, restocking and reintroduction plans should carefully consider the genetic background of the individuals.

  9. Genetic data provide evidence for wind-mediated transmission of highly pathogenic avian influenza.

    PubMed

    Ypma, Rolf J F; Jonges, Marcel; Bataille, Arnaud; Stegeman, Arjan; Koch, Guus; van Boven, Michiel; Koopmans, Marion; van Ballegooijen, W Marijn; Wallinga, Jacco

    2013-03-01

    Outbreaks of highly pathogenic avian influenza in poultry can cause severe economic damage and represent a public health threat. Development of efficient containment measures requires an understanding of how these influenza viruses are transmitted between farms. However, the actual mechanisms of interfarm transmission are largely unknown. Dispersal of infectious material by wind has been suggested, but never demonstrated, as a possible cause of transmission between farms. Here we provide statistical evidence that the direction of spread of avian influenza A(H7N7) is correlated with the direction of wind at date of infection. Using detailed genetic and epidemiological data, we found the direction of spread by reconstructing the transmission tree for a large outbreak in the Netherlands in 2003. We conservatively estimate the contribution of a possible wind-mediated mechanism to the total amount of spread during this outbreak to be around 18%.

  10. Genetic variation in the α1A-adrenergic receptor and phenylephrine-mediated venoconstriction.

    PubMed

    Adefurin, A; Ghimire, L V; Kohli, U; Muszkat, M; Sofowora, G G; Li, C; Paranjape, S Y; Stein, C M; Kurnik, D

    2015-08-01

    There is large interindividual variability and ethnic differences in phenylephrine-mediated vasoconstriction. We tested the hypothesis that genetic variation in ADRA1A, the α1A adrenergic receptor gene, contributes to the variability and ethnic differences. We measured local dorsal hand vein responses to increasing doses of phenylephrine in 64 Caucasians and 42 African-Americans and genotyped for 32 ADRA1A single nucleotide polymorphisms. The ED50 ranged from 11 to 5442 ng min(-1), and the Emax ranged from 13.5-100%. The rs574647 variant was associated with a trend towards lower logED50 in each race and in the combined cohort (P=0.008). In addition, rs1079078 was associated with a trend to higher logED50 in each race and in the combined cohort (P=0.011). Neither variant accounted for the ethnic differences in response. None of the ADRA1A haplotypes was associated with the outcomes. In conclusion, ADRA1A variants do not contribute substantially to the marked interindividual variability or ethnic differences in phenylephrine-mediated venoconstriction.

  11. Feral Cat Globetrotters: genetic traces of historical human-mediated dispersal.

    PubMed

    Koch, Katrin; Algar, Dave; Schwenk, Klaus

    2016-08-01

    Endemic species on islands are highly susceptible to local extinction, in particular if they are exposed to invasive species. Invasive predators, such as feral cats, have been introduced to islands around the world, causing major losses in local biodiversity. In order to control and manage invasive species successfully, information about source populations and level of gene flow is essential. Here, we investigate the origin of feral cats of Hawaiian and Australian islands to verify their European ancestry and a potential pattern of isolation by distance. We analyzed the genetic structure and diversity of feral cats from eleven islands as well as samples from Malaysia and Europe using mitochondrial DNA (ND5 and ND6 regions) and microsatellite DNA data. Our results suggest an overall European origin of Hawaiian cats with no pattern of isolation by distance between Australian, Malaysian, and Hawaiian populations. Instead, we found low levels of genetic differentiation between samples from Tasman Island, Lana'i, Kaho'olawe, Cocos (Keeling) Island, and Asia. As these populations are separated by up to 10,000 kilometers, we assume an extensive passive dispersal event along global maritime trade routes in the beginning of the 19th century, connecting Australian, Asian, and Hawaiian islands. Thus, islands populations, which are characterized by low levels of current gene flow, represent valuable sources of information on historical, human-mediated global dispersal patterns of feral cats.

  12. Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

    PubMed

    Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

    2012-11-07

    Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%−0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

  13. Ecological dynamics and complex interactions of Agrobacterium megaplasmids

    PubMed Central

    Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

  14. Adaptive genetic variation mediates bottom-up and top-down control in an aquatic ecosystem.

    PubMed

    Rudman, Seth M; Rodriguez-Cabal, Mariano A; Stier, Adrian; Sato, Takuya; Heavyside, Julian; El-Sabaawi, Rana W; Crutsinger, Gregory M

    2015-08-07

    Research in eco-evolutionary dynamics and community genetics has demonstrated that variation within a species can have strong impacts on associated communities and ecosystem processes. Yet, these studies have centred around individual focal species and at single trophic levels, ignoring the role of phenotypic variation in multiple taxa within an ecosystem. Given the ubiquitous nature of local adaptation, and thus intraspecific variation, we sought to understand how combinations of intraspecific variation in multiple species within an ecosystem impacts its ecology. Using two species that co-occur and demonstrate adaptation to their natal environments, black cottonwood (Populus trichocarpa) and three-spined stickleback (Gasterosteus aculeatus), we investigated the effects of intraspecific phenotypic variation on both top-down and bottom-up forces using a large-scale aquatic mesocosm experiment. Black cottonwood genotypes exhibit genetic variation in their productivity and consequently their leaf litter subsidies to the aquatic system, which mediates the strength of top-down effects from stickleback on prey abundances. Abundances of four common invertebrate prey species and available phosphorous, the most critically limiting nutrient in freshwater systems, are dictated by the interaction between genetic variation in cottonwood productivity and stickleback morphology. These interactive effects fit with ecological theory on the relationship between productivity and top-down control and are comparable in strength to the effects of predator addition. Our results illustrate that intraspecific variation, which can evolve rapidly, is an under-appreciated driver of community structure and ecosystem function, demonstrating that a multi-trophic perspective is essential to understanding the role of evolution in structuring ecological patterns.

  15. TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

    PubMed

    Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    2014-01-01

    Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

  16. ipt Gene transformation in petunia by an Agrobacterium mediated method.

    PubMed

    Bai, L J; Ye, C J; Lu, J Y; Yang, D E; Xue, H; Pan, Y; Cao, P X; Wang, B; Liu, M

    2009-01-01

    To prevent leaf senescence of petunia, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of 35S promoter and introduced into petunia. PCR analysis showed an expected 0.5 Kb fragment of ipt gene in transgenic petunia. RT-PCR analysis indicated the expression of ipt gene in the transgenic lines. Leaves from transgenic plants remained green and healthy in normal culture condition, while the non-transformed plants turned to yellow. Transgenic plants showed a reduction in height and smaller leaf sizes. In transgenic lines, the internodes were shorter, and the roots grew slower than the non-transformed plants.

  17. Developmental Mediation of Genetic Variation in Response to the Fast Track Prevention Program

    PubMed Central

    Albert, Dustin; Belsky, Daniel W.; Crowley, D. Max; Bates, John E.; Pettit, Gregory S.; Lansford, Jennifer E.; Dick, Danielle; Dodge, Kenneth A.

    2015-01-01

    We conducted a developmental analysis of genetic moderation of the effect of the Fast Track intervention on adult externalizing psychopathology. The Fast Track intervention enrolled 891 children at high risk to develop externalizing behavior problems when they were in kindergarten. Half of the enrolled children were randomly assigned to receive 10 years of treatment with a range of services and resources provided to the children and their families and the other half to usual care (controls). We previously showed that the effect of the Fast Track intervention on participants’ risk of externalizing psychopathology at age 25 years was moderated by a variant in the Glucocorticoid Receptor Gene (NR3C1). Children who carried copies of the A-allele of the single-nucleotide polymorphism rs10482672 had the highest risk of externalizing psychopathology if they were in the control arm of the trial and the lowest risk of externalizing psychopathology if they were in the treatment arm. In this study, we test a developmental hypothesis about the origins of this for-better-and-for-worse gene-by-intervention interaction (GxI): That the observed GxI effect on adult psychopathology is mediated by the proximal impact of intervention on childhood externalizing problems and adolescent substance use and delinquency. We analyzed longitudinal data tracking the 270 European-American children in the Fast Track RCT with available genetic information (129 intervention children and 141 control-group peers, 69% male) from kindergarten through age 25 years. Results show that the same pattern of “for-better-and-for-worse” susceptibility to intervention observed at the age-25 follow-up was evident already during childhood. At the elementary school follow-ups and at the middle/high-school follow-ups, rs10482672 predicted better adjustment among children receiving the Fast Track intervention, and worse adjustment among children in the control condition. In turn, these proximal GxI effects

  18. Agrobacterium rhizogenes: Transformed root cultures for the study of polyacetylene metabolism and biosynthesis

    SciTech Connect

    Marchant, Y.Y.

    1988-02-01

    Biologically active polyacetylenes are produced at low levels by the roots of members of the Coreopsidinae subtribe in the Asteraceae. Ten taxa of Coreopsis and Bidens were tranformed with Agrobacterium rhizogenes Strain A/sub 4/ and hairy root cultures established. These cultures grew rapidly and produced the same arrays of polyacetylenes as intact roots. The use of transformed roots for the study of polyacetylene biosynthesis is described in this paper. The engineering of plants with resistance to herbicides is now a practical reality because there are economic, intellectual and environmental incentives for using recombinant DNA technology in crop improvement programs, and because the biochemical and genetic basis for herbicide resistance is a simple trait conferred by a single gene. The transformation of plants with genes conferring resistance to insects or disease is more daunting, however, as biologically active secondary metabolites such as some alkaloids are typically products of multienzyme reactions. Photoactive polyacetylenes are probably plant defense chemicals and they are derived by a sequence of desaturation steps from oleic acid, which occurs ubiquitously in higher plants. Although the acetylene pathway may encompass as many genetic messages as those for morphine biosynthesis, it is likley that the genes controlling the biosynthesis of polyacetylenes may be isolated, identified in the near future and transferred via Agrobacterium to economically important plants susceptible to pathogen attack. 58 refs., 4 figs., 3 tabs.

  19. Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens

    PubMed Central

    Kado, Clarence I.

    2014-01-01

    The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

  20. Association Between Genetic Traits for Immune-Mediated Diseases and Alzheimer Disease

    PubMed Central

    Yokoyama, Jennifer S.; Wang, Yunpeng; Schork, Andrew J.; Thompson, Wesley K.; Karch, Celeste M.; Cruchaga, Carlos; McEvoy, Linda K.; Witoelar, Aree; Chen, Chi-Hua; Holland, Dominic; Brewer, James B.; Franke, Andre; Dillon, William P.; Wilson, David M.; Mukherjee, Pratik; Hess, Christopher P.; Miller, Zachary; Bonham, Luke W.; Shen, Jeffrey; Rabinovici, Gil D.; Rosen, Howard J.; Miller, Bruce L.; Hyman, Bradley T.; Schellenberg, Gerard D.; Karlsen, Tom H.; Andreassen, Ole A.; Dale, Anders M.; Desikan, Rahul S.

    2016-01-01

    IMPORTANCE Late-onset Alzheimer disease (AD), the most common form of dementia, places a large burden on families and society. Although epidemiological and clinical evidence suggests a relationship between inflammation and AD, their relationship is not well understood and could have implications for treatment and prevention strategies. OBJECTIVE To determine whether a subset of genes involved with increased risk of inflammation are also associated with increased risk for AD. DESIGN, SETTING, AND PARTICIPANTS In a genetic epidemiology study conducted in July 2015, we systematically investigated genetic overlap between AD (International Genomics of Alzheimer’s Project stage 1) and Crohn disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, and psoriasis using summary data from genome-wide association studies at multiple academic clinical research centers. P values and odds ratios from genome-wide association studies of more than 100 000 individuals were from previous comparisons of patients vs respective control cohorts. Diagnosis for each disorder was previously established for the parent study using consensus criteria. MAIN OUTCOMES AND MEASURES The primary outcome was the pleiotropic (conjunction) false discovery rate P value. Follow-up for candidate variants included neuritic plaque and neurofibrillary tangle pathology; longitudinal Alzheimer’s Disease Assessment Scale cognitive subscale scores as a measure of cognitive dysfunction (Alzheimer’s Disease Neuroimaging Initiative); and gene expression in AD vs control brains (Gene Expression Omnibus data). RESULTS Eight single-nucleotide polymorphisms (false discovery rate P < .05) were associated with both AD and immune-mediated diseases. Of these, rs2516049 (closest gene HLA-DRB5; conjunction false discovery rate P = .04 for AD and psoriasis, 5.37 × 10−5 for AD, and 6.03 × 10−15 for psoriasis) and rs12570088 (closest gene IPMK; conjunction false discovery rate P = .009 for

  1. Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators.

    PubMed

    Wong, Stanley S C; Kotera, Ippei; Mills, Evan; Suzuki, Hiroshi; Truong, Kevin

    2012-01-01

    While genetically encoded Ca(2+) indicators (GECIs) allow Ca(2+) imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca(2+) imaging, it had a, diminished Ca(2+) response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca(2+) response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca(2+) signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.

  2. Genetic dissection of pheromone processing reveals main olfactory system-mediated social behaviors in mice.

    PubMed

    Matsuo, Tomohiko; Hattori, Tatsuya; Asaba, Akari; Inoue, Naokazu; Kanomata, Nobuhiro; Kikusui, Takefumi; Kobayakawa, Reiko; Kobayakawa, Ko

    2015-01-20

    Most mammals have two major olfactory subsystems: the main olfactory system (MOS) and vomeronasal system (VNS). It is now widely accepted that the range of pheromones that control social behaviors are processed by both the VNS and the MOS. However, the functional contributions of each subsystem in social behavior remain unclear. To genetically dissociate the MOS and VNS functions, we established two conditional knockout mouse lines that led to either loss-of-function in the entire MOS or in the dorsal MOS. Mice with whole-MOS loss-of-function displayed severe defects in active sniffing and poor survival through the neonatal period. In contrast, when loss-of-function was confined to the dorsal MOB, sniffing behavior, pheromone recognition, and VNS activity were maintained. However, defects in a wide spectrum of social behaviors were observed: attraction to female urine and the accompanying ultrasonic vocalizations, chemoinvestigatory preference, aggression, maternal behaviors, and risk-assessment behaviors in response to an alarm pheromone. Functional dissociation of pheromone detection and pheromonal induction of behaviors showed the anterior olfactory nucleus (AON)-regulated social behaviors downstream from the MOS. Lesion analysis and neural activation mapping showed pheromonal activation in multiple amygdaloid and hypothalamic nuclei, important regions for the expression of social behavior, was dependent on MOS and AON functions. Identification of the MOS-AON-mediated pheromone pathway may provide insights into pheromone signaling in animals that do not possess a functional VNS, including humans.

  3. MicroRNA-mediated gene regulation: potential applications for plant genetic engineering.

    PubMed

    Zhou, Man; Luo, Hong

    2013-09-01

    Food security is one of the most important issues challenging the world today. Any strategies to solve this problem must include increasing crop yields and quality. MicroRNA-based genetic modification technology (miRNA-based GM tech) can be one of the most promising solutions that contribute to agricultural productivity directly by developing superior crop cultivars with enhanced biotic and abiotic stress tolerance and increased biomass yields. Indirectly, the technology may increase usage of marginal soils and decrease pesticide use, among other benefits. This review highlights the most recent progress of transgenic studies utilizing various miRNAs and their targets for plant trait modifications, and analyzes the potential of miRNA-mediated gene regulation for use in crop improvement. Strategies for manipulating miRNAs and their targets in transgenic plants including constitutive, stress-induced, or tissue-specific expression of miRNAs or their targets, RNA interference, expressing miRNA-resistant target genes, artificial target mimic and artificial miRNAs were discussed. We also discussed potential risks of utilizing miRNA-based GM tech. In general, miRNAs and their targets not only provide an invaluable source of novel transgenes, but also inspire the development of several new GM strategies, allowing advances in breeding novel crop cultivars with agronomically useful characteristics.

  4. Psychopathic personality traits and environmental contexts: Differential correlates, gender differences, and genetic mediation.

    PubMed

    Hicks, Brian M; Carlson, Marie D; Blonigen, Daniel M; Patrick, Christopher J; Iacono, William G; Mgue, Matt

    2012-07-01

    Theorists have speculated that primary psychopathy (or Factor 1 affective-interpersonal features) is prominently heritable whereas secondary psychopathy (or Factor 2 social deviance) is more environmentally determined. We tested this differential heritability hypothesis using a large adolescent twin sample. Trait-based proxies of primary and secondary psychopathic tendencies were assessed using Multidimensional Personality Questionnaire (MPQ) estimates of Fearless Dominance and Impulsive Antisociality, respectively. The environmental contexts of family, school, peers, and stressful life events were assessed using multiple raters and methods. Consistent with prior research, MPQ Impulsive Antisociality was robustly associated with each environmental risk factor, and these associations were significantly greater than those for MPQ Fearless Dominance. However, MPQ Fearless Dominance and Impulsive Antisociality exhibited similar heritability, and genetic effects mediated the associations between MPQ Impulsive Antisociality and the environmental measures. Results were largely consistent across male and female twins. We conclude that gene-environment correlations rather than main effects of genes and environments account for the differential environmental correlates of primary and secondary psychopathy.

  5. Rhizobium pusense is the main human pathogen in the genus Agrobacterium/Rhizobium.

    PubMed

    Aujoulat, F; Marchandin, H; Zorgniotti, I; Masnou, A; Jumas-Bilak, E

    2015-05-01

    Rhizobium pusense was recently described after isolation from the rhizosphere of chickpea. Multilocus sequence-based analysis of clinical isolates identified as Agrobacterium (Rhizobium) radiobacter demonstrated that R. pusense is the main human pathogen within Agrobacterium (Rhizobium) spp. Clinical microbiology of Agrobacterium (Rhizobium) should be considered in the light of recent taxonomic changes.

  6. Two-way chemical signaling in Agrobacterium-plant interactions.

    PubMed Central

    Winans, S C

    1992-01-01

    The discovery in 1977 that Agrobacterium species can transfer a discrete segment of oncogenic DNA (T-DNA) to the genome of host plant cells has stimulated an intense interest in the molecular biology underlying these plant-microbe associations. This attention in turn has resulted in a series of insights about the biology of these organisms that continue to accumulate at an ever-increasing rate. This excitement was due in part to the notion that this unprecedented interkingdom DNA transfer could be exploited to create transgenic plants containing foreign genes of scientific or commercial importance. In the course of these discoveries, Agrobacterium became one of the best available models for studying the molecular interactions between bacteria and higher organisms. One extensively studied aspect of this association concerns the exchange of chemical signals between Agrobacterium spp. and host plants. Agrobacterium spp. can recognize no fewer than five classes of low-molecular-weight compounds released from plants, and other classes probably await discovery. The most widely studied of these are phenolic compounds, which stimulate the transcription of the genes needed for infection. Other compounds include specific monosaccharides and acidic environments which potentiate vir gene induction, acidic polysaccharides which induce one or more chromosomal genes, and a family of compounds called opines which are released from tumorous plant cells to the bacteria as nutrient sources. Agrobacterium spp. in return release a variety of chemical compounds to plants. The best understood is the transferred DNA itself, which contains genes that in various ways upset the balance of phytohormones, ultimately causing neoplastic cell proliferation. In addition to transferring DNA, some Agrobacterium strains directly secrete phytohormones. Finally, at least some strains release a pectinase, which degrades a component of plant cell walls. PMID:1579105

  7. Seed-mediated gene flow promotes genetic diversity of weedy rice within populations: implications for weed management.

    PubMed

    He, Zhuoxian; Jiang, Xiaoqi; Ratnasekera, Disna; Grassi, Fabrizio; Perera, Udugahapattuwage; Lu, Bao-Rong

    2014-01-01

    Increased infestation of weedy rice-a noxious agricultural pest has caused significant reduction of grain yield of cultivated rice (Oryza sativa) worldwide. Knowledge on genetic diversity and structure of weedy rice populations will facilitate the design of effective methods to control this weed by tracing its origins and dispersal patterns in a given region. To generate such knowledge, we studied genetic diversity and structure of 21 weedy rice populations from Sri Lanka based on 23 selected microsatellite (SSR) loci. Results indicated an exceptionally high level of within-population genetic diversity (He = 0.62) and limited among-population differentiation (Fst = 0.17) for this predominantly self-pollinating weed. UPGMA analysis showed a loose genetic affinity of the weedy rice populations in relation to their geographical locations, and no obvious genetic structure among populations across the country. This phenomenon was associated with the considerable amount of gene flow between populations. Limited admixture from STRUCTURE analyses suggested a very low level of hybridization (pollen-mediated gene flow) between populations. The abundant within-population genetic diversity coupled with limited population genetic structure and differentiation is likely caused by the considerable seed-mediated gene flow of weedy rice along with the long-distance exchange of farmer-saved rice seeds between weedy-rice contaminated regions in Sri Lanka. In addition to other effective weed management strategies, promoting the application of certified rice seeds with no weedy rice contamination should be the immediate action to significantly reduce the proliferation and infestation of this weed in rice ecosystems in countries with similar rice farming styles as in Sri Lanka.

  8. Metallothionein-mediated neuroprotection in genetically engineered mouse models of Parkinson's disease

    PubMed Central

    Ebadi, Manuchair; Brown-Borg, Holly; Refaey, Hesham El; Singh, Brij B.; Garrett, Scott; Shavali, Shaik; Sharma, Sushil K.

    2013-01-01

    Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other sub-cortical nuclei associated with a widespread occurrence of Lewy bodies. The cause of cell death in Parkinson's disease is still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative and nitrative stresses have been proposed. We have studied controlwt (C57B1/6), metallothionein transgenic (MTtrans), metallothionein double gene knock (MTdko), α-synuclein knock out (α-synko), α-synuclein–metallothionein triple knock out (α-syn–MTtko), weaver mutant (wv/wv) mice, and Ames dwarf mice to examine the role of peroxynitrite in the etiopathogenesis of Parkinson's disease and aging. Although MTdko mice were genetically susceptible to 1, methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism, they did not exhibit any overt clinical symptoms of neurodegeneration and gross neuropathological changes as observed in wv/wv mice. Progressive neurodegenerative changes were associated with typical Parkinsonism in wv/wv mice. Neurodegenerative changes in wv/wv mice were observed primarily in the striatum, hippocampus and cerebellum. Various hallmarks of apoptosis including caspase-3, TNFα, NFκB, metallothioneins (MT-1, 2) and complex-1 nitration were increased; whereas glutathione, complex-1, ATP, and Ser(40)-phosphorylation of tyrosine hydroxylase, and striatal 18F-DOPA uptake were reduced in wv/wv mice as compared to other experimental genotypes. Striatal neurons of wv/wv mice exhibited age-dependent increase in dense cored intra-neuronal inclusions, cellular aggregation, proto-oncogenes (c-fos, c-jun, caspase-3, and GAPDH) induction, inter-nucleosomal DNA fragmentation, and neuro-apoptosis. MTtrans and α-Synko mice were genetically resistant to MPTP-Parkinsonism and Ames dwarf mice possessed significantly higher concentrations of striatal coenzyme Q10 and metallothioneins (MT 1, 2

  9. Rhizobia species: A Boon for "Plant Genetic Engineering".

    PubMed

    Patel, Urmi; Sinha, Sarika

    2011-10-01

    Since past three decades new discoveries in plant genetic engineering have shown remarkable potentials for crop improvement. Agrobacterium Ti plasmid based DNA transfer is no longer the only efficient way of introducing agronomically important genes into plants. Recent studies have explored a novel plant genetic engineering tool, Rhizobia sp., as an alternative to Agrobacterium, thereby expanding the choice of bacterial species in agricultural plant biotechnology. Rhizobia sp. serve as an open license source with no major restrictions in plant biotechnology and help broaden the spectrum for plant biotechnologists with respect to the use of gene transfer vehicles in plants. New efficient transgenic plants can be produced by transferring genes of interest using binary vector carrying Rhizobia sp. Studies focusing on the interactions of Rhizobia sp. with their hosts, for stable and transient transformation and expression of genes, could help in the development of an adequate gene transfer vehicle. Along with being biologically beneficial, it may also bring a new means for fast economic development of transgenic plants, thus giving rise to a new era in plant biotechnology, viz. "Rhizobia mediated transformation technology."

  10. Large Deletions in the pAtC58 Megaplasmid of Agrobacterium tumefaciens Can Confer Reduced Carriage Cost and Increased Expression of Virulence Genes

    PubMed Central

    Morton, Elise R.; Merritt, Peter M.; Bever, James D.; Fuqua, Clay

    2013-01-01

    The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115–194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids. PMID:23783172

  11. Agrobacterium tumefaciens-Induced Bacteraemia Does Not Lead to Reporter Gene Expression in Mouse Organs

    PubMed Central

    Petrunia, Igor V.; Frolova, Olga Y.; Komarova, Tatiana V.; Kiselev, Sergey L.; Citovsky, Vitaly; Dorokhov, Yuri L.

    2008-01-01

    Agrobacterium tumefaciens is the main plant biotechnology gene transfer tool with host range which can be extended to non-plant eukaryotic organisms under laboratory conditions. Known medical cases of Agrobacterium species isolation from bloodstream infections necessitate the assessment of biosafety-related risks of A. tumefaciens encounters with mammalian organisms. Here, we studied the survival of A. tumefaciens in bloodstream of mice injected with bacterial cultures. Bacterial titers of 108 CFU were detected in the blood of the injected animals up to two weeks after intravenous injection. Agrobacteria carrying Cauliflower mosaic virus (CaMV) 35S promoter-based constructs and isolated from the injected mice retained their capacity to promote green fluorescent protein (GFP) synthesis in Nicotiana benthamiana leaves. To examine whether or not the injected agrobacteria are able to express in mouse organs, we used an intron-containing GFP (GFPi) reporter driven either by a cytomegalovirus (CMV) promoter or by a CaMV 35S promoter. Western and northern blot analyses as well as RT-PCR analysis of liver, spleen and lung of mice injected with A. tumefaciens detected neither GFP protein nor its transcripts. Thus, bacteraemia induced in mice by A. tumefaciens does not lead to detectible levels of genetic transformation of mouse organs. PMID:18523638

  12. Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.

    PubMed

    Gabryszewski, Stanislaw J; Modchang, Charin; Musset, Lise; Chookajorn, Thanat; Fidock, David A

    2016-06-01

    The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.

  13. Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

    PubMed

    Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

    2013-11-01

    In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

  14. The limits of child effects: evidence for genetically mediated child effects on corporal punishment but not on physical maltreatment.

    PubMed

    Jaffee, Sara R; Caspi, Avshalom; Moffitt, Terrie E; Polo-Tomas, Monica; Price, Thomas S; Taylor, Alan

    2004-11-01

    Research on child effects has demonstrated that children's difficult and coercive behavior provokes harsh discipline from adults. Using a genetically sensitive design, the authors tested the limits of child effects on adult behavior that ranged from the normative (corporal punishment) to the nonnormative (physical maltreatment). The sample was a 1994-1995 nationally representative birth cohort of 1,116 twins and their families who participated in the Environmental Risk Longitudinal Study. Results showed that environmental factors accounted for most of the variation in corporal punishment and physical maltreatment. However, corporal punishment was genetically mediated in part, and the genetic factors that influenced corporal punishment were largely the same as those that influenced children's antisocial behavior, suggesting a child effect. The authors conclude that risk factors for maltreatment are less likely to reside within the child and more likely to reside in characteristics that differ between families.

  15. Genetics

    MedlinePlus

    ... Inheritance; Heterozygous; Inheritance patterns; Heredity and disease; Heritable; Genetic markers ... The chromosomes are made up of strands of genetic information called DNA. Each chromosome contains sections of ...

  16. [Agrobacterium rubi strains from blueberry plants are highly diverse].

    PubMed

    Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

    2014-01-01

    The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation.

  17. Complete Genome Sequence of Agrobacterium tumefaciens Ach5

    PubMed Central

    Huang, Ya-Yi; Cho, Shu-Ting; Lo, Wen-Sui; Wang, Yi-Chieh; Lai, Erh-Min

    2015-01-01

    Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease. The strain Ach5 was isolated from yarrow (Achillea ptarmica L.) and is the wild-type progenitor of other derived strains widely used for plant transformation. Here, we report the complete genome sequence of this bacterium. PMID:26044425

  18. Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work reports the draft genome of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, and is composed of 1 circular chromosome, the Ri virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild type strain cau...

  19. Reiterated DNA sequences in Rhizobium and Agrobacterium spp.

    PubMed Central

    Flores, M; González, V; Brom, S; Martínez, E; Piñero, D; Romero, D; Dávila, G; Palacios, R

    1987-01-01

    Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome. Images PMID:3450286

  20. Virulence of Agrobacterium tumefaciens strain A281 on legumes

    SciTech Connect

    Hood, E.E.; Fraley, R.T.; Chilton, M.D.

    1987-03-01

    This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an L,L-succinamopine strain. The authors tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

  1. Impact of biological amendments on Agrobacterium tumefaciens soil survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paradox, the primary walnut rootstock used in California, is susceptible to Agrobacterium tumefaciens, which causes crown gall. While A. tumefaciens is susceptible to commonly used fumigants such as methyl bromide (MeBr) and Telone-C35 (1,3-dichloropropene and chloropicrin), these fumigants also sig...

  2. Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants

    PubMed Central

    Circelli, Patrizia; Donini, Marcello; Villani, Maria Elena; Benvenuto, Eugenio

    2010-01-01

    We have recently described an efficient transient expression system mediated by Agrobacterium tumefaciens for the production of HIV-1 Nef protein in Nicotiana benthamiana plants. In order to enhance the yield of recombinant protein we assayed the effect of three gene-silencing viral suppressor proteins (P25 of Potato Virus X, P19 of Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef expression levels. Results demonstrated that AMCV-P19 gave the highest Nef yield (1.3% of total soluble protein) and that this effect was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms. Here we report additional data on the production of different heterologous proteins including human immunoglobulin heavy and light chains and a virus coat protein that demonstrate the robustness of this co-agroinfiltration expression system boosted by the AMCV-P19 gene-silencing suppressor. PMID:21326930

  3. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    PubMed

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

  4. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus.

    PubMed

    Yuan, Yao-Wu; Sagawa, Janelle M; Young, Riane C; Christensen, Brian J; Bradshaw, Harvey D

    2013-05-01

    Prezygotic barriers play a major role in the evolution of reproductive isolation, which is a prerequisite for speciation. However, despite considerable progress in identifying genes and mutations responsible for postzygotic isolation, little is known about the genetic and molecular basis underlying prezygotic barriers. The bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis represent a classic example of pollinator-mediated prezygotic isolation between two sister species in sympatry. Flower color differences resulting from both carotenoid and anthocyanin pigments contribute to pollinator discrimination between the two species in nature. Through fine-scale genetic mapping, site-directed mutagenesis, and transgenic experiments, we demonstrate that a single-repeat R3 MYB repressor, ROSE INTENSITY1 (ROI1), is the causal gene underlying a major quantitative trait locus (QTL) with the largest effect on anthocyanin concentration and that cis-regulatory change rather than coding DNA mutations cause the allelic difference between M. lewisii and M. cardinalis. Together with the genomic resources and stable transgenic tools developed here, these results suggest that Mimulus is an excellent platform for studying the genetics of pollinator-mediated reproductive isolation and the molecular basis of morphological evolution at the most fundamental level-gene by gene, mutation by mutation.

  5. Relaxation of herbivore-mediated selection drives the evolution of genetic covariances between plant competitive and defense traits.

    PubMed

    Uesugi, Akane; Connallon, Tim; Kessler, André; Monro, Keyne

    2017-04-10

    Insect herbivores are important mediators of selection on traits that impact plant defense against herbivory and competitive ability. Although recent experiments demonstrate a central role for herbivory in driving rapid evolution of defense and competition-mediating traits, whether and how herbivory shapes heritable variation in these traits remains poorly understood. Here, we evaluate the structure and evolutionary stability of the G matrix for plant metabolites that are involved in defense and allelopathy in the tall goldenrod, Solidago altissima. We show that G has evolutionarily diverged between experimentally-replicated populations that evolved in the presence versus the absence of ambient herbivory, providing direct evidence for the evolution of G by natural selection. Specifically, evolution in an herbivore-free habitat altered the orientation of G, revealing a negative genetic covariation between defense- and competition-related metabolites that is typically masked in herbivore-exposed populations. Our results may be explained by predictions of classical quantitative genetic theory, as well as the theory of acquisition-allocation trade-offs. The study provides compelling evidence that herbivory drives the evolution of plant genetic architecture. This article is protected by copyright. All rights reserved.

  6. Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium

    PubMed Central

    Slater, Steven; Setubal, João C.; Houmiel, Kathryn; Sun, Jian; Kaul, Rajinder; Goldman, Barry S.; Farrand, Stephen K.; Almeida, Nalvo; Burr, Thomas; Nester, Eugene; Rhoads, David M.; Kadoi, Ryosuke; Ostheimer, Trucian; Pride, Nicole; Sabo, Allison; Henry, Erin; Telepak, Erin; Cromes, Lindsey; Harkleroad, Alana; Oliphant, Louis; Pratt-Szegila, Phil; Welch, Roy; Wood, Derek

    2013-01-01

    Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium. PMID:23241979

  7. Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore

    PubMed Central

    Penyalver, Ramón; Oger, Philippe; López, María M.; Farrand, Stephen K.

    2001-01-01

    Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing

  8. The Baldwin effect and genetic assimilation: revisiting two mechanisms of evolutionary change mediated by phenotypic plasticity.

    PubMed

    Crispo, Erika

    2007-11-01

    Two different, but related, evolutionary theories pertaining to phenotypic plasticity were proposed by James Mark Baldwin and Conrad Hal Waddington. Unfortunately, these theories are often confused with one another. Baldwin's notion of organic selection posits that plasticity influences whether an individual will survive in a new environment, thus dictating the course of future evolution. Heritable variations can then be selected upon to direct phenotypic evolution (i.e., "orthoplasy"). The combination of these two processes (organic selection and orthoplasy) is now commonly referred to as the "Baldwin effect." Alternately, Waddington's genetic assimilation is a process whereby an environmentally induced phenotype, or "acquired character," becomes canalized through selection acting upon the developmental system. Genetic accommodation is a modern term used to describe the process of heritable changes that occur in response to a novel induction. Genetic accommodation is a key component of the Baldwin effect, and genetic assimilation is a type of genetic accommodation. I here define both the Baldwin effect and genetic assimilation in terms of genetic accommodation, describe cases in which either should occur in nature, and propose that each could play a role in evolutionary diversification.

  9. Aggression as a mediator of genetic contributions to the association between negative parent-child relationships and adolescent antisocial behavior.

    PubMed

    Narusyte, Jurgita; Andershed, Anna-Karin; Neiderhiser, Jenae M; Lichtenstein, Paul

    2007-03-01

    Previous research suggests that the association between conflictual parent-child relationships and maladjustment among adolescents is influenced by genetic effects emanating from the adolescents. In this study, we examined whether these effects are mediated by childhood aggression. The data come from the Twin study of CHild and Adolescent Development (TCHAD), a Swedish longitudinal study including 1,314 twin pairs followed from age 13-14 to 16-17. Early adolescent aggression, parental criticism, and delinquency in later adolescence were rated by parents and children at different time points. Multivariate genetic structural equation models were used to estimate genetic and environmental influences on these constructs and on their covariation. The results showed that approximately half of the genetic contribution to the association between parental criticism and delinquency was explained by early adolescent aggression. It suggests that aggression in children evokes negative parenting, which in turn influences adolescent antisocial behavior. The mechanism proposed by these findings is consistent with evocative gene-environment correlation.

  10. A combination of developmental plasticity, parental effects, and genetic differentiation mediates divergences in life history traits between dung beetle populations.

    PubMed

    Beckers, Oliver M; Anderson, Wendy; Moczek, Armin P

    2015-01-01

    The dung beetle, Onthophagus taurus, was introduced <50 years ago from its native Mediterranean range into Western Australia (WA) and the Eastern United States (EUS). The intensity of intra- and interspecific competition for dung as a breeding resource is substantially higher in WA. First, we tested whether differential resource competition in the two exotic ranges is associated with divergences in life history traits, which impact on resource use. We predicted that high levels of resource competition in WA should favor females that produce brood balls more efficiently and of altered size, and produce offspring more readily when a breeding opportunity arises. Furthermore, we predicted that larvae from WA populations may have evolved more efficient development and thus exhibit higher eclosion success, shorter development time, and altered body size under standardized conditions. Second, we examined the likely developmental mechanisms underlying these divergences, that is, genetic differentiation, developmental plasticity, or parental effects in a common garden experiment. Field-collected EUS and WA populations significantly differed, as predicted, in most of the traits examined. However, these differences are facilitated by a complex combination of proximate mechanisms. Developmental plasticity and (grand) parental effects mediated differences related to reproductive performance, whereas genetic differentiation mediated differences in the duration of larval development. Our study highlights that population divergences can be the product of a patchwork of proximate mechanisms, with each mechanism adjusting different traits in a way that the resulting composite phenotype may be better suited to its competitive environment.

  11. A mathematical model gives insights into nutritional and genetic aspects of folate-mediated one-carbon metabolism.

    PubMed

    Reed, Michael C; Nijhout, H Frederik; Neuhouser, Marian L; Gregory, Jesse F; Shane, Barry; James, S Jill; Boynton, Alanna; Ulrich, Cornelia M

    2006-10-01

    Impaired folate-mediated 1-carbon metabolism has been linked to multiple disease outcomes. A better understanding of the nutritional and genetic influences on this complex biochemical pathway is needed to comprehend their impact on human health. To this end, we created a mathematical model of folate-mediated 1-carbon metabolism. The model uses published data on folate enzyme kinetics and regulatory mechanisms to simulate the impact of genetic and nutritional variation on critical aspects of the pathway. We found that the model predictions match experimental data, while providing novel insights into pathway kinetics. Our primary observations were as follows: 1) the inverse association between folate and homocysteine is strongest at very low folate concentrations, but there is no association at high folate concentrations; 2) the DNA methylation reaction rate is relatively insensitive to changes in folate pool size; and 3) as folate concentrations become very high, enzyme velocities decrease. With regard to polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), the modeling predicts that decrease MTHFR activity reduces concentrations of S-adenosylmethionine and 5-methyltetrahydrofolate, as well as DNA methylation, while modestly increasing S-adenosylhomocysteine and homocysteine concentrations and thymidine or purine synthesis. Decreased folate together with a simulated vitamin B-12 deficiency results in decreases in DNA methylation and purine and thymidine synthesis. Decreased MTHFR activity superimposed on the B-12 deficiency appears to reverse the declines in purine and thymidine synthesis. These mathematical simulations of folate-mediated 1-carbon metabolism provide a cost-efficient approach to in silico experimentation that can complement and help guide laboratory studies.

  12. Copy-choice recombination by reverse transcriptases: Reshuffling of genetic markers mediated by RNA chaperones

    PubMed Central

    Negroni, Matteo; Buc, Henri

    2000-01-01

    Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses. PMID:10829081

  13. Genetics and molecular biology of siderophore-mediated iron transport in bacteria.

    PubMed Central

    Crosa, J H

    1989-01-01

    The possession of specialized iron transport systems may be crucial for bacteria to override the iron limitation imposed by the host or the environment. One of the most commonly found strategies evolved by microorganisms is the production of siderophores, low-molecular-weight iron chelators that have very high constants of association for their complexes with iron. Thus, siderophores act as extracellular solubilizing agents for iron from minerals or organic compounds, such as transferrin and lactoferrin in the host vertebrate, under conditions of iron limitation. Transport of iron into the cell cytosol is mediated by specific membrane receptor and transport systems which recognize the iron-siderophore complexes. In this review I have analyzed in detail three siderophore-mediated iron uptake systems: the plasmid-encoded anguibactin system of Vibrio anguillarum, the aerobactin-mediated iron assimilation system present in the pColV-K30 plasmid and in the chromosomes of many enteric bacteria, and the chromosomally encoded enterobactin iron uptake system, found in Escherichia coli, Shigella spp., Salmonella spp., and other members of the family Enterobacteriaceae. The siderophore systems encoded by Pseudomonas aeruginosa, namely, pyochelin and pyoverdin, as well as the siderophore amonabactin, specified by Aeromonas hydrophila, are also discussed. The potential role of siderophore-mediated systems as virulence determinants in the specific host-bacteria interaction leading to disease is also analyzed with respect to the influence of these systems in the expression of other factors, such as toxins, in the bacterial virulence repertoire. PMID:2531838

  14. SES and CHAOS as Environmental Mediators of Cognitive Ability: A Longitudinal Genetic Analysis

    ERIC Educational Resources Information Center

    Hart, Sara A.; Petrill, Stephen A.; Deckard, Kirby Deater; Thompson, Lee A.

    2007-01-01

    This study examined shared environmental influences on the longitudinal stability of general cognitive ability, as mediated by socioeconomic status and chaos in the home, using 287 pairs of elementary school-age twins drawn from the Western Reserve Reading Project (WRRP). General cognitive ability was evaluated at two annual assessments using the…

  15. Genetic and epigenetic determinants mediate proneness of oncogene breakpoint sites for involvement in TCR translocations.

    PubMed

    Larmonie, N S D; van der Spek, A; Bogers, A J J C; van Dongen, J J M; Langerak, A W

    2014-03-01

    T-cell receptor (TCR) translocations are a genetic hallmark of T-cell acute lymphoblastic leukemia and lead to juxtaposition of oncogene and TCR loci. Oncogene loci become involved in translocations because they are accessible to the V(D)J recombination machinery. Such accessibility is predicted at cryptic recombination signal sequence (cRSS) sites ('Type 1') as well as other sites that are subject to DNA double-strand breaks (DSBs) ('Type 2') during early stages of thymocyte development. As chromatin accessibility markers have not been analyzed in the context of TCR-associated translocations, various genetic and epigenetic determinants of LMO2, TAL1 and TLX1 translocation breakpoint (BP) sites and BP cluster regions (BCRs) were examined in human thymocytes to establish DSB proneness and heterogeneity of BP site involvement in TCR translocations. Our data show that DSBs in BCRs are primarily induced in the presence of a genetic element of sequence vulnerability (cRSSs, transposable elements), whereas breaks at single BP sites lacking such elements are more likely induced by chance or perhaps because of patient-specific genetic vulnerability. Vulnerability to obtain DSBs is increased by features that determine chromatin organization, such as methylation status and nucleosome occupancy, although at different levels at different BP sites.

  16. An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

    PubMed Central

    Klauser, Benedikt; Hartig, Jörg S.

    2013-01-01

    An important requirement for achieving many goals of synthetic biology is the availability of a large repertoire of reprogrammable genetic switches and appropriate transmitter molecules. In addition to engineering genetic switches, the interconnection of individual switches becomes increasingly important for the construction of more complex genetic networks. In particular, RNA-based switches of gene expression have become a powerful tool to post-transcriptionally program genetic circuits. RNAs used for regulatory purposes have the advantage to transmit, sense, process and execute information. We have recently used the hammerhead ribozyme to control translation initiation in a small molecule-dependent fashion. In addition, riboregulators have been constructed in which a small RNA acts as transmitter molecule to control translation of a target mRNA. In this study, we combine both concepts and redesign the hammerhead ribozyme to sense small trans-acting RNAs (taRNAs) as input molecules resulting in repression of translation initiation in Escherichia coli. Importantly, our ribozyme-based expression platform is compatible with previously reported artificial taRNAs, which were reported to act as inducers of gene expression. In addition, we provide several insights into key requirements of riboregulatory systems, including the influences of varying transcriptional induction of the taRNA and mRNA transcripts, 5′-processing of taRNAs, as well as altering the secondary structure of the taRNA. In conclusion, we introduce an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits. PMID:23585277

  17. A Genetically Encoded Alkyne Directs Palladium-Mediated Protein Labeling on Live Mammalian Cell Surface

    PubMed Central

    2015-01-01

    The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry. PMID:25347611

  18. The genetic mediation of individual differences in sensitivity to pain and its inhibition

    PubMed Central

    Mogil, Jeffrey S.

    1999-01-01

    The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a combination of “top-down” and “bottom-up” strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents. PMID:10393892

  19. Plasticity and genetic adaptation mediate amphibian and reptile responses to climate change.

    PubMed

    Urban, Mark C; Richardson, Jonathan L; Freidenfelds, Nicole A

    2014-01-01

    Phenotypic plasticity and genetic adaptation are predicted to mitigate some of the negative biotic consequences of climate change. Here, we evaluate evidence for plastic and evolutionary responses to climate variation in amphibians and reptiles via a literature review and meta-analysis. We included studies that either document phenotypic changes through time or space. Plasticity had a clear and ubiquitous role in promoting phenotypic changes in response to climate variation. For adaptive evolution, we found no direct evidence for evolution of amphibians or reptiles in response to climate change over time. However, we found many studies that documented adaptive responses to climate along spatial gradients. Plasticity provided a mixture of adaptive and maladaptive responses to climate change, highlighting that plasticity frequently, but not always, could ameliorate climate change. Based on our review, we advocate for more experiments that survey genetic changes through time in response to climate change. Overall, plastic and genetic variation in amphibians and reptiles could buffer some of the formidable threats from climate change, but large uncertainties remain owing to limited data.

  20. Plasticity and genetic adaptation mediate amphibian and reptile responses to climate change

    PubMed Central

    Urban, Mark C; Richardson, Jonathan L; Freidenfelds, Nicole A

    2014-01-01

    Phenotypic plasticity and genetic adaptation are predicted to mitigate some of the negative biotic consequences of climate change. Here, we evaluate evidence for plastic and evolutionary responses to climate variation in amphibians and reptiles via a literature review and meta-analysis. We included studies that either document phenotypic changes through time or space. Plasticity had a clear and ubiquitous role in promoting phenotypic changes in response to climate variation. For adaptive evolution, we found no direct evidence for evolution of amphibians or reptiles in response to climate change over time. However, we found many studies that documented adaptive responses to climate along spatial gradients. Plasticity provided a mixture of adaptive and maladaptive responses to climate change, highlighting that plasticity frequently, but not always, could ameliorate climate change. Based on our review, we advocate for more experiments that survey genetic changes through time in response to climate change. Overall, plastic and genetic variation in amphibians and reptiles could buffer some of the formidable threats from climate change, but large uncertainties remain owing to limited data. PMID:24454550

  1. A chemical genetics approach reveals H,K-ATPase-mediated membrane voltage is required for planarian head regeneration.

    PubMed

    Beane, Wendy S; Morokuma, Junji; Adams, Dany S; Levin, Michael

    2011-01-28

    Biophysical signaling is required for both embryonic polarity and regenerative outgrowth. Exploiting endogenous ion transport for regenerative therapies will require direct regulation of membrane voltage. Here, we develop a pharmacological method to target ion transporters, uncovering a role for membrane voltage as a key regulator of anterior polarity in regenerating planaria. Utilizing the highly specific inhibitor, SCH-28080, our data reveal that H(+),K(+)-ATPase-mediated membrane depolarization is essential for anterior gene expression and brain induction. H(+),K(+)-ATPase-independent manipulation of membrane potential with ivermectin confirms that depolarization drives head formation, even at posterior-facing wounds. Using this chemical genetics approach, we demonstrate that membrane voltage controls head-versus-tail identity during planarian regeneration. Our data suggest well-characterized drugs (already approved for human use) might be exploited to control adult stem cell-driven pattern formation during the regeneration of complex structures.

  2. Molecular Genetic Tools and Techniques for Marchantia polymorpha Research.

    PubMed

    Ishizaki, Kimitsune; Nishihama, Ryuichi; Yamato, Katsuyuki T; Kohchi, Takayuki

    2016-02-01

    Liverworts occupy a basal position in the evolution of land plants, and are a key group to address a wide variety of questions in plant biology. Marchantia polymorpha is a common, easily cultivated, dioecious liverwort species, and is emerging as an experimental model organism. The haploid gametophytic generation dominates the diploid sporophytic generation in its life cycle. Genetically homogeneous lines in the gametophyte generation can be established easily and propagated through asexual reproduction, which aids genetic and biochemical experiments. Owing to its dioecy, male and female sexual organs are formed in separate individuals, which enables crossing in a fully controlled manner. Reproductive growth can be induced at the desired times under laboratory conditions, which helps genetic analysis. The developmental process from a single-celled spore to a multicellular body can be observed directly in detail. As a model organism, molecular techniques for M. polymorpha are well developed; for example, simple and efficient protocols of Agrobacterium-mediated transformation have been established. Based on them, various strategies for molecular genetics, such as introduction of reporter constructs, overexpression, gene silencing and targeted gene modification, are available. Herein, we describe the technologies and resources for reverse and forward genetics in M. polymorpha, which offer an excellent experimental platform to study the evolution and diversity of regulatory systems in land plants.

  3. Transposon-Assisted Genetic Engineering with Mos1-Mediated Single-Copy Insertion (MosSCI).

    PubMed

    Frøkjær-Jensen, Christian

    2015-01-01

    Transgenesis in model organisms is necessary to determine the function, expression, and subcellular localization of gene products. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extrachromosomal arrays but transgenes in arrays are mosaic, over-expressed in some tissues and silenced in the germline. Here, a method to insert a transgene into a specific genomic location called Mos1-mediated single-copy insertion (MosSCI) is described. Single-copy insertion allows transgene expression at levels that approximate endogenous gene expression as well as expression in the germline.

  4. Genetic structure and seed-mediated dispersal rates of an endangered shrub in a fragmented landscape: a case study for Juniperus communis in northwestern Europe

    PubMed Central

    2011-01-01

    Background Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity. Results Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability. Conclusions In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run. PMID:21859457

  5. A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

    PubMed Central

    Feirer, Nathan; Xu, Jing; Allen, Kylie D.; Koestler, Benjamin J.; Bruger, Eric L.; Waters, Christopher M.; White, Robert H.

    2015-01-01

    ABSTRACT The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins. PMID:26126849

  6. Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

    PubMed

    Chu, Weihua; Vattem, Dhiraj A; Maitin, Vatsala; Barnes, Mary B; McLean, Robert J C

    2011-01-01

    In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

  7. Genetic partitioning of interleukin-6 signalling in mice dissociates Stat3 from Smad3-mediated lung fibrosis

    PubMed Central

    O'Donoghue, Robert J J; Knight, Darryl A; Richards, Carl D; Prêle, Cecilia M; Lau, Hui Ling; Jarnicki, Andrew G; Jones, Jessica; Bozinovski, Steven; Vlahos, Ross; Thiem, Stefan; McKenzie, Brent S; Wang, Bo; Stumbles, Philip; Laurent, Geoffrey J; McAnulty, Robin J; Rose-John, Stefan; Zhu, Hong Jian; Anderson, Gary P; Ernst, Matthias R; Mutsaers, Steven E

    2012-01-01

    Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-β signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130757F mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130757F;µMT−/− compound mutant mice, but fibrosis still occurred in their Smad3−/− counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-β/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis. PMID:22684844

  8. Loop-mediated isothermal amplification: rapid visual and real-time methods for detection of genetically modified crops.

    PubMed

    Randhawa, Gurinder Jit; Singh, Monika; Morisset, Dany; Sood, Payal; Zel, Jana

    2013-11-27

    A rapid, reliable, and sensitive loop-mediated isothermal amplification (LAMP) system was developed for screening of genetically modified organisms (GMOs). The optimized LAMP assays using designed primers target commonly employed promoters, i.e., Cauliflower Mosaic Virus 35S (P-35S) and Figwort Mosaic Virus promoter (P-FMV), and marker genes, i.e., aminoglycoside 3'-adenyltransferase (aadA), neomycin phosphotransferase II (nptII), and β-glucuronidase (uidA). The specificity and performance of the end-point and real-time LAMP assays were confirmed using eight genetically modified (GM) cotton events on four detection systems, employing two chemistries. LAMP assays on the isothermal real-time system were found to be most sensitive, detecting up to four target copies, within 35 min. The LAMP assays herein presented using alternate detection systems can be effectively utilized for rapid and cost-effective screening of the GM status of a sample, irrespective of the crop species or GM trait. These assays coupled with a fast and simple DNA extraction method may further facilitate on-site GMO screening.

  9. Linear amplification mediated PCR--localization of genetic elements and characterization of unknown flanking DNA.

    PubMed

    Gabriel, Richard; Kutschera, Ina; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred

    2014-06-25

    Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3'- and 5'- sequences adjacent to the integrated lentiviral vector.

  10. Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.

    PubMed

    Fradin, Emilie F; Zhang, Zhao; Juarez Ayala, Juan C; Castroverde, Christian D M; Nazar, Ross N; Robb, Jane; Liu, Chun-Ming; Thomma, Bart P H J

    2009-05-01

    Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The Verticillium genus includes vascular wilt pathogens with a wide host range. Although V. longisporum infects various hosts belonging to the Cruciferaceae, V. dahliae and V. albo-atrum cause vascular wilt diseases in over 200 dicotyledonous species, including economically important crops. A locus responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum has been cloned from tomato (Solanum lycopersicum) only. This locus, known as Ve, comprises two closely linked inversely oriented genes, Ve1 and Ve2, that encode cell surface receptor proteins of the extracellular leucine-rich repeat receptor-like protein class of disease resistance proteins. Here, we show that Ve1, but not Ve2, provides resistance in tomato against race 1 strains of V. dahliae and V. albo-atrum and not against race 2 strains. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 is shown to require both EDS1 and NDR1. In addition, NRC1, ACIF, MEK2, and SERK3/BAK1 also act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the receptor-like protein class of resistance proteins.

  11. Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

    PubMed

    Anuradha, T Swathi; Jami, S K; Datla, R S; Kirti, P B

    2006-06-01

    We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

  12. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    PubMed

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

  13. Genetically and functionally defined NTS to PBN brain circuits mediating anorexia

    PubMed Central

    Roman, Carolyn W.; Derkach, Victor A.; Palmiter, Richard D.

    2016-01-01

    The central nervous system controls food consumption to maintain metabolic homoeostasis. In response to a meal, visceral signals from the gut activate neurons in the nucleus of the solitary tract (NTS) via the vagus nerve. These NTS neurons then excite brain regions known to mediate feeding behaviour, such as the lateral parabrachial nucleus (PBN). We previously described a neural circuit for appetite suppression involving calcitonin gene-related protein (CGRP)-expressing PBN (CGRPPBN) neurons; however, the molecular identity of the inputs to these neurons was not established. Here we identify cholecystokinin (CCK) and noradrenergic, dopamine β-hydroxylase (DBH)-expressing NTS neurons as two separate populations that directly excite CGRPPBN neurons. When these NTS neurons are activated using optogenetic or chemogenetic methods, food intake decreases and with chronic stimulation mice lose body weight. Our optogenetic results reveal that CCK and DBH neurons in the NTS directly engage CGRPPBN neurons to promote anorexia. PMID:27301688

  14. [Genetic transformation of OSISAP1 gene to onion (Allium cepa L.) mediated by amicroprojectile bombardment].

    PubMed

    Xu, Qi-Jiang; Cui, Cheng-Ri

    2007-06-01

    Microprojectile bombardment-mediated transformation method has been developed for onion (Allium cepa L.) using embryogenic calli, induced from stem discs, as target tissue. Zinc-finger protein gene OSISAP1 (Oryza sative subspecies indica stress-associated protein gene) was introduced into the open-pollinated onion cultivar (subs.) 'HG400B'. Bombardment parameters were optimized as: the pressure is 1,100 psi, the distance is 6 cm, two times, the ratio of mass between plasmid DNA and golden particles is 1:320. An efficient microprojectile bombardment-mediated transformation system of onion (Allium cepa L.) callus has been established. The binary vector used carried the nptII gene for kanamycin resistance and the GUS reporter gene. Transgenic cultures were screened for their ability to express the GUS reporter gene and to grow in the presence of kanamycin (150 mg/L). Transient expression of GUS reporter gene was observed through histochemical staining of embryogenic callus transformed by microprojectile bombardment. The putative transgenic plants were analysed at the molecular level using PCR, southern hybridization, and RT-PCR. The results confirmed that the OSISAP1 gene was integrated as one copy into the genome of onion and expression. Transgenic plants were produced efficiently with a transformation frequency of about 10%. Test of salinity-alkali stress showed that sodium chloride and sodium bicarbonate at 200 mmol/L effectively killed non-transgenic plants within 1 week of irrigation, while the transgenic plants were completely unaffected by salinity of 400 mmol/L. So transformation with the OSISAP1 gene raised the salinity-alkali-tolerance of the transgenic plants to a high level.

  15. Antisense-Mediated RNA Targeting: Versatile and Expedient Genetic Manipulation in the Brain

    PubMed Central

    Zalachoras, Ioannis; Evers, Melvin M.; van Roon-Mom, Willeke M. C.; Aartsma-Rus, Annemieke M.; Meijer, Onno C.

    2011-01-01

    A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized. PMID:21811437

  16. Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

    PubMed

    Swain, S S; Sahu, L; Pal, A; Barik, D P; Pradhan, C; Chand, P K

    2012-02-01

    Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD(660) ≅ 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.

  17. Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

    PubMed

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

    2014-06-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

  18. Genetic Mediators of Neurocognitive Outcomes in Survivors of Childhood Acute Lymphoblastic Leukemia

    PubMed Central

    Krull, Kevin R.; Bhojwani, Deepa; Conklin, Heather M.; Pei, Deqing; Cheng, Cheng; Reddick, Wilburn E.; Sandlund, John T.; Pui, Ching-Hon

    2013-01-01

    Purpose Survivors of childhood acute lymphoblastic leukemia (ALL) are at increased risk for neurocognitive problems, with significant interindividual variability in outcome. This study examined genetic polymorphisms associated with variability in neurocognitive outcome. Patients and Methods Neurocognitive outcomes were evaluated at the end of therapy in 243 survivors treated on an institutional protocol featuring risk-adapted chemotherapy without prophylactic cranial irradiation. Polymorphisms in genes related to pharmacokinetics or pharmacodynamics of antileukemic agents, drug metabolism, oxidative stress, and attention problems in noncancer populations were examined as predictors of outcome, using multiple general linear models and controlling for age at diagnosis, sex, race, and treatment intensity. Results Compared with national norms, the cohort demonstrated significantly higher rates of problems on direct assessment of sustained attention (P = .01) and on parent ratings of attention problems (P = .02). Children with the A2756G polymorphism in methionine synthase (MS) were more likely to demonstrate deficits in attentiveness (P = .03) and response speed (P = .02), whereas those with various polymorphisms in glutathione S-transferase demonstrated increased performance variability (P = .01) and reduced attentiveness (P = .003). Polymorphisms in monoamine oxidase (T1460CA) were associated with increased attention variability (P = .03). Parent-reported attention problems were more common in children with the Cys112Arg polymorphism in apoliopoprotein E4 (P = .01). Conclusion These results are consistent with our previous report of association between attention problems and MS in an independent cohort of long-term survivors of childhood ALL treated with chemotherapy only. The results also raise the possibility of an impact from genetic predispositions related to oxidative stress and CNS integrity. PMID:23650422

  19. Simple screening strategy with only water bath needed for the identification of insect-resistant genetically modified rice.

    PubMed

    Zhang, Fang; Wang, Liu; Wang, Rui; Ying, Yibin; Wu, Jian

    2015-02-03

    An informative, with simple instrument needed, rapid and easily updated strategy for the identification of insect-resistant genetically modified (GM) rice has been described. Such strategy is based on a parallel series of loop-mediated isothermal amplification (LAMP) reactions targeting the rice endogenous gene sucrose phosphate synthase (Sps), the top two most frequently used genetic elements (Agrobacterium tumefaciens nopaline synthase terminator (Nos) and Cauliflower mosaic virus 35S promoter (CaMV35S)), and an insect-resistant specific gene (Cry1Ac) and detected visually by phosphate ion (Pi)-induced coloration reaction. After a logical judgment of visible readouts has been obtained, three popular insect-resistant GM rice events in China can be successfully identified within 35 min, using either microwell strips or paper bases.

  20. Genetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Capsicum represents one of several well characterized Solanaceous genera. A wealth of classical and molecular genetics research is available for the genus. Information gleaned from its cultivated relatives, tomato and potato, provide further insight for basic and applied studies. Early ...

  1. Genetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maintaining genetic variation in wild populations of Arctic organisms is fundamental to the long-term persistence of high latitude biodiversity. Variability is important because it provides options for species to respond to changing environmental conditions and novel challenges such as emerging path...

  2. Fate of Agrobacterium radiobacter K84 in the environment.

    PubMed Central

    Stockwell, V O; Moore, L W; Loper, J E

    1993-01-01

    Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue. PMID:8357247

  3. T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

    PubMed

    van Kregten, Maartje; de Pater, Sylvia; Romeijn, Ron; van Schendel, Robin; Hooykaas, Paul J J; Tijsterman, Marcel

    2016-10-31

    Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown(1). Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

  4. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop.

    PubMed

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve; Kreuze, Jan F

    2015-05-05

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world's arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

  5. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

    PubMed Central

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve

    2015-01-01

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops. PMID:25902487

  6. Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity.

    PubMed

    Angoa-Pérez, Mariana; Kane, Michael J; Briggs, Denise I; Sykes, Catherine E; Shah, Mrudang M; Francescutti, Dina M; Rosenberg, David R; Thomas, David M; Kuhn, Donald M

    2012-06-01

    Neuropsychiatric disorders characterized by behavioral disinhibition, including disorders of compulsivity (e.g. obsessive-compulsive disorder; OCD) and impulse-control (e.g. impulsive aggression), are severe, highly prevalent and chronically disabling. Treatment options for these diseases are extremely limited. The pathophysiological bases of disorders of behavioral disinhibition are poorly understood but it has been suggested that serotonin dysfunction may play a role. Mice lacking the gene encoding brain tryptophan hydroxylase 2 (Tph2-/-), the initial and rate-limiting enzyme in the synthesis of serotonin, were tested in numerous behavioral assays that are well known for their utility in modeling human neuropsychiatric diseases. Mice lacking Tph2 (and brain 5HT) show intense compulsive and impulsive behaviors to include extreme aggression. The impulsivity is motor in form and not cognitive because Tph2-/- mice show normal acquisition and reversal learning on a spatial learning task. Restoration of 5HT levels by treatment of Tph2-/- mice with its immediate precursor 5-hydroxytryptophan attenuated compulsive and impulsive-aggressive behaviors. Surprisingly, in Tph2-/- mice, the lack of 5HT was not associated with anxiety-like behaviors. The results indicate that 5HT mediates behavioral disinhibition in the mammalian brain independent of anxiogenesis.

  7. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    PubMed

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  8. Temperament and Externalizing Behavior as Mediators of Genetic Risk on Adolescent Substance Use

    PubMed Central

    Trucco, Elisa M.; Hicks, Brian M.; Villafuerte, Sandra; Nigg, Joel; Burmeister, Margit; Zucker, Robert A.

    2016-01-01

    Understanding how specific genes contribute to risk for addiction remains challenging. This study tests whether childhood temperament and externalizing behavior in early adolescence account for a portion of the association between specific genetic variants and substance use problems in late adolescence. The sample consisted of 487 adolescents from the Michigan Longitudinal Study, a high-risk sample (70.2% male, 81.7% European American ancestry). Polymorphisms across serotonergic (SLC6A4, 5-HTTLPR), dopaminergic (DRD4, u-VNTR), noradrenergic (SLC6A2, rs36021), and GABAergic (GABRA2, rs279858; GABRA6, rs3811995) genes were examined given prior support for associations with temperament, externalizing behavior, and substance use problems. The temperament traits behavioral control and resiliency were assessed using interviewer ratings (ages 9-11), and externalizing behavior (ages 12-14) was assessed using teacher ratings. Self-reported substance use outcomes (ages 15-17) included maximum alcoholic beverages consumed in 24 hours, and frequency of past year cigarette and marijuana use. Behavioral control, resiliency, and externalizing behavior accounted for the associations between polymorphisms in noradrenergic and GABAergic genes and substance use in late adolescence. Individual differences in emotional coping and behavioral regulation represent non-specific neurobiological underpinnings for an externalizing pathway to addiction. PMID:26845260

  9. Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

    PubMed

    Mou, Haiwei; Moore, Jill; Malonia, Sunil K; Li, Yingxiang; Ozata, Deniz M; Hough, Soren; Song, Chun-Qing; Smith, Jordan L; Fischer, Andrew; Weng, Zhiping; Green, Michael R; Xue, Wen

    2017-04-04

    Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

  10. Sexual dimorphism in xenobiotic genetic variants-mediated risk for Parkinson's disease.

    PubMed

    Kumudini, Nadella; Uma, Addepally; Naushad, Shaik Mohammad; Mridula, Rukmini; Borgohain, Rupam; Kutala, Vijay Kumar

    2014-06-01

    The vulnerability of dopaminergic neurons to environmental exposures in sporadic Parkinson's disease (PD) has been attributed to altered detoxification by xenobiotic metabolizing genes. Hence, we investigated the influence of genetic polymorphisms in xenobiotic metabolic pathway (CYP1A1 m1, CYP1A1 m2, CYP1A1 m4, COMT p.H108L, GSTT1, and GSTM1) on the susceptibility to PD. We used PCR-RFLP for CYP1A1 and COMT genotyping; multiplex-PCR for GSTT1 and GSTM1 deletion analysis; and spectrophotometric methods to evaluate the oxidative stress markers. Results showed association of CYP1A1 m1 (OR: 2.38, 95 % CI: 1.76-3.22) and COMT p.H108L (OR: 2.08 95 % CI: 1.56-2.77) polymorphisms with risk for PD. Male patients carrying combination of COMT p.H108L and CYP1A1 m1 variant alleles showed an early onset of the disease. There was a significant increase in oxidative stress makers such as malondialdehyde and protein carbonyls; and decrease in glutathione levels in PD cases compared to controls (P < 0.05). To conclude, CYP1A1 m1, COMT p.H108L polymorphisms were associated with PD risk, and sexual dimorphism was observed in these associations.

  11. Striatal Dopamine Mediates the Interface between Motivational and Cognitive Control in Humans: Evidence from Genetic Imaging

    PubMed Central

    Aarts, Esther; Roelofs, Ardi; Franke, Barbara; Rijpkema, Mark; Fernández, Guillén; Helmich, Rick C; Cools, Roshan

    2010-01-01

    Dopamine has been hypothesized to provide the basis for the interaction between motivational and cognitive control. However, there is no evidence for this hypothesis in humans. We fill this gap by using fMRI, a novel behavioral paradigm and a common polymorphism in the DAT1 gene (SLC6A3). Carriers of the 9-repeat (9R) allele of a 40 base pair repeat polymorphism in the 3′ untranslated region of DAT1, associated with high striatal dopamine, showed greater activity in the ventromedial striatum during reward anticipation than homozygotes for the 10-repeat allele, replicating previous genetic imaging studies. The crucial novel finding is that 9R carriers also exhibited a greater influence of anticipated reward on switch costs, as well as greater activity in the dorsomedial striatum during task switching in anticipation of high reward relative to low reward. These data establish a crucial role for human striatal dopamine in the modulation of cognitive flexibility by reward anticipation, thus, elucidating the neurochemical mechanism of the interaction between motivation and cognitive control. PMID:20463658

  12. Striatal dopamine mediates the interface between motivational and cognitive control in humans: evidence from genetic imaging.

    PubMed

    Aarts, Esther; Roelofs, Ardi; Franke, Barbara; Rijpkema, Mark; Fernández, Guillén; Helmich, Rick C; Cools, Roshan

    2010-08-01

    Dopamine has been hypothesized to provide the basis for the interaction between motivational and cognitive control. However, there is no evidence for this hypothesis in humans. We fill this gap by using fMRI, a novel behavioral paradigm and a common polymorphism in the DAT1 gene (SLC6A3). Carriers of the 9-repeat (9R) allele of a 40 base pair repeat polymorphism in the 3' untranslated region of DAT1, associated with high striatal dopamine, showed greater activity in the ventromedial striatum during reward anticipation than homozygotes for the 10-repeat allele, replicating previous genetic imaging studies. The crucial novel finding is that 9R carriers also exhibited a greater influence of anticipated reward on switch costs, as well as greater activity in the dorsomedial striatum during task switching in anticipation of high reward relative to low reward. These data establish a crucial role for human striatal dopamine in the modulation of cognitive flexibility by reward anticipation, thus, elucidating the neurochemical mechanism of the interaction between motivation and cognitive control.

  13. Dorsal and ventral stream mediated visual processing in genetic subtypes of Prader-Willi syndrome.

    PubMed

    Woodcock, Kate A; Humphreys, Glyn W; Oliver, Chris

    2009-10-01

    Previous work has suggested that there are specific deficits in dorsal stream processing in a variety of developmental disorders. Prader-Willi syndrome (PWS) is associated with two main genetic subtypes, deletion and disomy. Relative strengths in visual processing are shown in PWS, although these strengths may be specific to the deletion subtype. We investigated visual processing in PWS using an adapted Simon task which contrasted location (dorsal stream) and shape identity (ventral stream) tasks. Compared to a group of typically developing children, children with PWS deletion showed a greater degree of impairment in the dorsal stream task than in the ventral stream task, a pattern similar to that shown in a group of boys with Fragile-X syndrome. When matched on a measure of non-verbal ability, children with PWS disomy showed the opposite pattern with better performance in the location compared to the shape task, although these task performance asymmetries may have been linked to executive control processes. It is proposed that children with PWS deletion show a relative strength in visual processing in the ventral stream along with a specific deficit in dorsal stream processing. In contrast, children with PWS disomy show neither effect.

  14. Carnitine supplementation has protective and detrimental effects in Saccharomyces cerevisiae that are genetically mediated.

    PubMed

    Franken, Jaco; Bauer, Florian F

    2010-05-01

    l-Carnitine plays a well-documented role in eukaryotic energy homeostasis by acting as a shuttling molecule for activated acyl residues across intracellular membranes. This activity, supported by carnitine acyl-transferases and transporters, is referred to as the carnitine shuttle. However, several pleiotropic and often beneficial effects of carnitine in humans have been reported that appear to be unrelated to shuttling activity, but little conclusive evidence regarding molecular mechanisms exists. We have recently demonstrated a role of carnitine, independent of the carnitine shuttle, in yeast stress protection. Here, we show that carnitine specifically protects against oxidative stress caused by H(2)O(2) and the superoxide-generating agent menadione. Surprisingly, carnitine has a detrimental effect on survival when combined with thiol-modifying agents. Central elements of the oxidative stress response, specifically the transcription factors Yap1p and Skn7p, are shown to be required for carnitine's protective effect, but several downstream effectors are dispensable. A DNA microarray-based analysis identifies Cyc3p, a cytochrome c heme lyase, as being important for carnitine's impact during oxidative stress. These findings establish a direct genetic link to a carnitine-related phenotype that is independent of the shuttle system and suggests that Saccharomyces cerevisiae should provide a useful model for further elucidation of carnitine's physiological roles.

  15. Agrobacterium rhizogenes GALLS Protein Substitutes for Agrobacterium tumefaciens Single-Stranded DNA-Binding Protein VirE2

    PubMed Central

    Hodges, Larry D.; Cuperus, Josh; Ream, Walt

    2004-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2. PMID:15126468

  16. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    NASA Astrophysics Data System (ADS)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  17. Spatial and Temporal Assessment of Pollen- and Seed-Mediated Gene Flow from Genetically Engineered Plum Prunus domestica

    PubMed Central

    Scorza, Ralph; Kriss, Alissa B.; Callahan, Ann M.; Webb, Kevin; Demuth, Mark; Gottwald, Tim

    2013-01-01

    Pollen flow from a 0.46 ha plot of genetically engineered (GE) Prunus domestica located in West Virginia, USA was evaluated from 2000–2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera) were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree) to 0.033–0.017% at longer distances (384–998 m). Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards. PMID:24098374

  18. Spatial and temporal assessment of pollen- and seed-mediated gene flow from genetically engineered plum Prunus domestica.

    PubMed

    Scorza, Ralph; Kriss, Alissa B; Callahan, Ann M; Webb, Kevin; Demuth, Mark; Gottwald, Tim

    2013-01-01

    Pollen flow from a 0.46 ha plot of genetically engineered (GE) Prunus domestica located in West Virginia, USA was evaluated from 2000-2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera) were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree) to 0.033-0.017% at longer distances (384-998 m). Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards.

  19. Exploring genetic moderators and epigenetic mediators of contextual and family effects: From Gene × Environment to epigenetics.

    PubMed

    Beach, Steven R H; Brody, Gene H; Barton, Allen W; Philibert, Robert A

    2016-11-01

    In the current manuscript, we provide an overview of a research program at the University of Georgia's Center for Family Research designed to expand upon rapid and ongoing developments in the fields of genetics and epigenetics. By placing those developments in the context of translational research on family and community determinants of health and well-being among rural African Americans, we hope to identify novel, modifiable environments and biological processes. In the first section of the article, we review our earlier work on genotypic variation effects on the association between family context and mental and physical health outcomes as well as differential responses to family-based intervention. We then transition to discuss our more recent research on the association of family and community environments with epigenetic processes. In this second section of the article, we begin by briefly reviewing terminology and basic considerations before describing evidence that early environments may influence epigenetic motifs that potentially serve as mediators of long-term effects of early family and community environments on longer term health outcomes. We also provide evidence that genotype may sometimes influence epigenetic outcomes. Finally, we describe our recent efforts to use genome-wide characterization of epigenetic patterns to better understand the biological impact of protective parenting on long-term shifts in inflammatory processes and its potential implications for young adult health. As will be clear, research on epigenetics as a mediator of the connections between family/community processes and a range of health outcomes is still in its infancy, but the potential to develop important insights regarding mechanisms linking modifiable environments to biological processes and long-term health outcomes already is coming into view.

  20. Development of Genetically Flexible Mouse Models of Sarcoma Using RCAS-TVA Mediated Gene Delivery

    PubMed Central

    Kabaroff, Leah; Gupta, Amar; Menezes, Serena; Babichev, Yael; Kandel, Rita C.; Swallow, Carol J.; Dickson, Brendan C.; Gladdy, Rebecca A.

    2014-01-01

    Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (p16/p19−/−) resulted in sarcoma in 30% of p16/p19−/−xN-tva mice with a median latency of 23 weeks (range 8–40 weeks). A similar incidence occurred in p16/p19−/−xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. p16/p19−/−xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research. PMID:24733554

  1. Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene.

    PubMed Central

    Heidekamp, F; Dirkse, W G; Hille, J; van Ormondt, H

    1983-01-01

    The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant cells. The nucleotide sequence of the tmr gene displays a continuous open reading frame specifying a polypeptide chain of 240 amino acids. The 5'- terminus of the polyadenylated tmr mRNA isolated from octopine tobacco tumor cell lines was determined by nuclease S1 mapping. The nucleotide sequence 5'-TATAAAA-3', which sequence is identical to the canonical "TATA" box, was found 29 nucleotides upstream from the major initiation site for RNA synthesis. Two potential polyadenylation signals 5'-AATAAA-3' were found at 207 and 275 nucleotides downstream from the TAG stopcodon of the tmr gene. A comparison was made of nucleotide stretches, involved in transcription control of T-DNA genes. Images PMID:6312414

  2. Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

    SciTech Connect

    Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

    2009-06-03

    N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

  3. Identification of Genetic Modules Mediating the Jekyll and Hyde Interaction of Dinoroseobacter shibae with the Dinoflagellate Prorocentrum minimum

    PubMed Central

    Wang, Hui; Tomasch, Jürgen; Michael, Victoria; Bhuju, Sabin; Jarek, Michael; Petersen, Jörn; Wagner-Döbler, Irene

    2015-01-01

    The co-cultivation of the alphaproteobacterium Dinoroseobacter shibae with the dinoflagellate Prorocentrum minimum is characterized by a mutualistic phase followed by a pathogenic phase in which the bacterium kills aging algae. Thus it resembles the “Jekyll-and-Hyde” interaction that has been proposed for other algae and Roseobacter. Here, we identified key genetic components of this interaction. Analysis of the transcriptome of D. shibae in co-culture with P. minimum revealed growth phase dependent changes in the expression of quorum sensing, the CtrA phosphorelay, and flagella biosynthesis genes. Deletion of the histidine kinase gene cckA which is part of the CtrA phosphorelay or the flagella genes fliC or flgK resulted in complete lack of growth stimulation of P. minimum in co-culture with the D. shibae mutants. By contrast, pathogenicity was entirely dependent on one of the extrachromosomal elements of D. shibae, the 191 kb plasmid. The data show that flagella and the CtrA phosphorelay are required for establishing mutualism and prove a cell density dependent killing effect of D. shibae on P. minimum which is mediated by an unknown factor encoded on the 191 kb plasmid. PMID:26617596

  4. Matrilineal genetic structure and female-mediated gene flow in red grouse (Lagopus lagopus scoticus): an analysis using mitochondrial DNA.

    PubMed

    Piertney, S B; MacColl, A D; Bacon, P J; Racey, P A; Lambin, X; Dallas, J F

    2000-02-01

    DNA sequence variation at the hypervariable 5' end of the mitochondrial control region was examined in 247 individuals to detect genetic divergence among 14 populations of red grouse (Lagopus lagopus scoticus) in northeastern Scotland. Ten haplotypes were resolved, several of which were shared among populations. Analysis of molecular variance, Nei's gamma ST, and a cladistic estimate of the amount of gene flow indicated a lack of overall population differentiation. Patterns of overall panmixia are in stark contrast to previous reports of localized subdivision among the same set of populations detected using hypervariable microsatellite markers. Because grouse cocks are territorial and show extreme natal philopatry and females are the dispersing sex, such discordance could be explained by sex-biased dispersal, with extensive female-mediated gene flow preventing mitochondrial DNA divergence. However, it is difficult to reconcile how effective dispersal of females would not homogenize both mitochondrial and nuclear structure simultaneously. We use a model that examines the spatial and temporal dynamics of diparentally and uniparentally inherited genes to show that, under realistic ecological scenarios and with specific differences in the dispersal of males and females, the local effective size of the nuclear genome can be less than that of the mitochondrial and the patterns of structuring we observe are meaningful.

  5. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

    PubMed Central

    Fujita, Naonobu; Huang, Wilson; Lin, Tzu-han; Groulx, Jean-Francois; Jean, Steve; Nguyen, Jen; Kuchitsu, Yoshihiko; Koyama-Honda, Ikuko; Mizushima, Noboru; Fukuda, Mitsunori; Kiger, Amy A

    2017-01-01

    Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23367.001 PMID:28063257

  6. AAV8-Mediated In Vivo Overexpression of miR-155 Enhances the Protective Capacity of Genetically Attenuated Malarial Parasites

    PubMed Central

    Hentzschel, Franziska; Hammerschmidt-Kamper, Christiane; Börner, Kathleen; Heiss, Kirsten; Knapp, Bettina; Sattler, Julia M; Kaderali, Lars; Castoldi, Mirco; Bindman, Julia G; Malato, Yann; Willenbring, Holger; Mueller, Ann-Kristin; Grimm, Dirk

    2014-01-01

    Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases. PMID:25189739

  7. Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

    PubMed

    Lidder, P; Johnson, M A; Sullivan, M L; Thompson, D M; Pérez-Amador, M A; Howard, C J; Green, P J

    2004-08-01

    mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

  8. Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes

    SciTech Connect

    Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. ); Ravelonandro, M. . Station de Pathologie Vegetale)

    1994-09-01

    Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).

  9. Succinoglycan production by solid-state fermentation with Agrobacterium tumefaciens.

    PubMed

    Stredansky, M; Conti, E

    1999-09-01

    Succinoglycan was produced by cultivating Agrobacterium tumefaciens on various solid substrates, including agar medium, spent malt grains, ivory nut shavings, and grated carrots, impregnated with a nutrient+ solution. Fermentations were performed on a laboratory scale, both under static conditions and with agitation, using bottles and a prototype horizontal bioreactor. Several fermentation parameters were examined and optimized, including carbon and nitrogen composition, water content and layer thickness of the substrate. The yields and rheological properties of the polymers obtained under different fermentation conditions were compared. The highest succinoglycan yield was achieved in static cultivation, reaching 42 g/l of impregnating solution, corresponding to 30 g/kg of wet substrate. The polymer production in the horizontal bioreactor was faster, but the final yield was lower (29 g/l of impregnating solution).

  10. Enhancement of Agrobacterium tumefaciens infectivity by mitomycin C.

    PubMed

    Heberlein, G T; Lippincott, J A

    1967-11-01

    The ability of Agrobacterium tumefaciens to induce pinto leaf tumors may be enhanced two- to threefold after treatment with mitomycin C. The enhancement may be obtained with either lethal or nonlethal concentrations. With 10-min treatments, an optimal response was obtained with 0.005 mug of mitomycin C per ml in the absence of any change in the number of viable cells. Both the tumor induction process and the tumors induced by treated cultures appear qualitatively the same as controls. To account for these results, the antibiotic must increase the proportion of viable cells that will subsequently initiate tumors. One, or at most a few, random lesions in the bacterial chromosome seem to be the necessary requirement for this promotion. At mitomycin concentrations of 1 and 5 mug/ml, the ability of A. tumefaciens to initiate tumors is rapidly lost, indicating that a fairly intact bacterial chromosome is one of the essentials for the tumor induction process.

  11. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

    PubMed

    Tollenaere, C; Bryja, J; Galan, M; Cadet, P; Deter, J; Chaval, Y; Berthier, K; Ribas Salvador, A; Voutilainen, L; Laakkonen, J; Henttonen, H; Cosson, J-F; Charbonnel, N

    2008-09-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

  12. Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

    PubMed Central

    Penalver, R.; Serra, M. T.; Duran-Vila, N.; Lopez, M. M.

    1996-01-01

    Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips. PMID:16535413

  13. Imaging Genetics

    ERIC Educational Resources Information Center

    Munoz, Karen E.; Hyde, Luke W.; Hariri, Ahmad R.

    2009-01-01

    Imaging genetics is an experimental strategy that integrates molecular genetics and neuroimaging technology to examine biological mechanisms that mediate differences in behavior and the risks for psychiatric disorder. The basic principles in imaging genetics and the development of the field are discussed.

  14. Unmasking host and microbial strategies in the Agrobacterium-plant defense tango

    PubMed Central

    Hwang, Elizabeth E.; Wang, Melinda B.; Bravo, Janis E.; Banta, Lois M.

    2015-01-01

    Coevolutionary forces drive adaptation of both plant-associated microbes and their hosts. Eloquently captured in the Red Queen Hypothesis, the complexity of each plant–pathogen relationship reflects escalating adversarial strategies, but also external biotic and abiotic pressures on both partners. Innate immune responses are triggered by highly conserved pathogen-associated molecular patterns, or PAMPs, that are harbingers of microbial presence. Upon cell surface receptor-mediated recognition of these pathogen-derived molecules, host plants mount a variety of physiological responses to limit pathogen survival and/or invasion. Successful pathogens often rely on secretion systems to translocate host-modulating effectors that subvert plant defenses, thereby increasing virulence. Host plants, in turn, have evolved to recognize these effectors, activating what has typically been characterized as a pathogen-specific form of immunity. Recent data support the notion that PAMP-triggered and effector-triggered defenses are complementary facets of a convergent, albeit differentially regulated, set of immune responses. This review highlights the key players in the plant’s recognition and signal transduction pathways, with a focus on the aspects that may limit Agrobacterium tumefaciens infection and the ways it might overcome those defenses. Recent advances in the field include a growing appreciation for the contributions of cytoskeletal dynamics and membrane trafficking to the regulation of these exquisitely tuned defenses. Pathogen counter-defenses frequently manipulate the interwoven hormonal pathways that mediate host responses. Emerging systems-level analyses include host physiological factors such as circadian cycling. The existing literature indicates that varying or even conflicting results from different labs may well be attributable to environmental factors including time of day of infection, temperature, and/or developmental stage of the host plant. PMID:25873923

  15. Slit/Robo-mediated axon guidance in Tribolium and Drosophila: divergent genetic programs build insect nervous systems

    PubMed Central

    Evans, Timothy A.; Bashaw, Greg J.

    2014-01-01

    As the complexity of animal nervous systems has increased during evolution, developmental control of neuronal connectivity has become increasingly refined. How has functional diversification within related axon guidance molecules contributed to the evolution of nervous systems? To address this question, we explore the evolution of functional diversity within the Roundabout (Robo) family of axon guidance receptors. In Drosophila, Robo and Robo2 promote midline repulsion, while Robo2 and Robo3 specify the position of longitudinal axon pathways. The Robo family has expanded by gene duplication in insects; robo2 and robo3 exist as distinct genes only within dipterans, while other insects, like the flour beetle Tribolium castaneum, retain an ancestral robo2/3 gene. Both Robos from Tribolium can mediate midline repulsion in Drosophila, but unlike the fly Robos cannot be down-regulated by Commissureless. The overall architecture and arrangement of longitudinal pathways are remarkably conserved in Tribolium, despite it having only two Robos. Loss of TcSlit causes midline collapse of axons in the beetle, a phenotype recapitulated by simultaneous knockdown of both Robos. Single gene knockdowns reveal that beetle Robos have specialized axon guidance functions: TcRobo is dedicated to midline repulsion, while TcRobo2/3 also regulates longitudinal pathway formation. TcRobo2/3 knockdown reproduces aspects of both Drosophila robo2 and robo3 mutants, suggesting that TcRobo2/3 has two functions that in Drosophila are divided between Robo2 and Robo3. The ability of Tribolium to organize longitudinal axons into three discrete medial-lateral zones with only two Robo receptors demonstrates that beetle and fly achieve equivalent developmental outcomes using divergent genetic programs. PMID:22245052

  16. Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders.

    PubMed

    Usdin, Karen; Hayward, Bruce E; Kumari, Daman; Lokanga, Rachel A; Sciascia, Nicholas; Zhao, Xiao-Nan

    2014-01-01

    The Fragile X-related disorders are a group of genetic conditions that include the neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS), the fertility disorder, Fragile X-associated primary ovarian insufficiency (FXPOI) and the intellectual disability, Fragile X syndrome (FXS). The pathology in all these diseases is related to the number of CGG/CCG-repeats in the 5' UTR of the Fragile X mental retardation 1 (FMR1) gene. The repeats are prone to continuous expansion and the increase in repeat number has paradoxical effects on gene expression increasing transcription on mid-sized alleles and decreasing it on longer ones. In some cases the repeats can simultaneously both increase FMR1 mRNA production and decrease the levels of the FMR1 gene product, Fragile X mental retardation 1 protein (FMRP). Since FXTAS and FXPOI result from the deleterious consequences of the expression of elevated levels of FMR1 mRNA and FXS is caused by an FMRP deficiency, the clinical picture is turning out to be more complex than once appreciated. Added complications result from the fact that increasing repeat numbers make the alleles somatically unstable. Thus many individuals have a complex mixture of different sized alleles in different cells. Furthermore, it has become apparent that the eponymous fragile site, once thought to be no more than a useful diagnostic criterion, may have clinical consequences for females who inherit chromosomes that express this site. This review will cover what is currently known about the mechanisms responsible for repeat instability, for the repeat-mediated epigenetic changes that affect expression of the FMR1 gene, and for chromosome fragility. It will also touch on what current and future options are for ameliorating some of these effects.

  17. Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders

    PubMed Central

    Usdin, Karen; Hayward, Bruce E.; Kumari, Daman; Lokanga, Rachel A.; Sciascia, Nicholas; Zhao, Xiao-Nan

    2014-01-01

    The Fragile X-related disorders are a group of genetic conditions that include the neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS), the fertility disorder, Fragile X-associated primary ovarian insufficiency (FXPOI) and the intellectual disability, Fragile X syndrome (FXS). The pathology in all these diseases is related to the number of CGG/CCG-repeats in the 5′ UTR of the Fragile X mental retardation 1 (FMR1) gene. The repeats are prone to continuous expansion and the increase in repeat number has paradoxical effects on gene expression increasing transcription on mid-sized alleles and decreasing it on longer ones. In some cases the repeats can simultaneously both increase FMR1 mRNA production and decrease the levels of the FMR1 gene product, Fragile X mental retardation 1 protein (FMRP). Since FXTAS and FXPOI result from the deleterious consequences of the expression of elevated levels of FMR1 mRNA and FXS is caused by an FMRP deficiency, the clinical picture is turning out to be more complex than once appreciated. Added complications result from the fact that increasing repeat numbers make the alleles somatically unstable. Thus many individuals have a complex mixture of different sized alleles in different cells. Furthermore, it has become apparent that the eponymous fragile site, once thought to be no more than a useful diagnostic criterion, may have clinical consequences for females who inherit chromosomes that express this site. This review will cover what is currently known about the mechanisms responsible for repeat instability, for the repeat-mediated epigenetic changes that affect expression of the FMR1 gene, and for chromosome fragility. It will also touch on what current and future options are for ameliorating some of these effects. PMID:25101111

  18. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish.

    PubMed

    Asakawa, Kazuhide; Suster, Maximiliano L; Mizusawa, Kanta; Nagayoshi, Saori; Kotani, Tomoya; Urasaki, Akihiro; Kishimoto, Yasuyuki; Hibi, Masahiko; Kawakami, Koichi

    2008-01-29

    Targeted gene expression is a powerful approach to study the function of genes and cells in vivo. In Drosophila, the P element-mediated Gal4-UAS method has been successfully used for this purpose. However, similar methods have not been established in vertebrates. Here we report the development of a targeted gene expression methodology in zebrafish based on the Tol2 transposable element and its application to the functional study of neural circuits. First, we developed gene trap and enhancer trap constructs carrying an engineered yeast Gal4 transcription activator (Gal4FF) and transgenic reporter fish carrying the GFP or the RFP gene downstream of the Gal4 recognition sequence (UAS) and showed that the Gal4FF can activate transcription through UAS in zebrafish. Second, by using this Gal4FF-UAS system, we performed large-scale screens and generated a large collection of fish lines that expressed Gal4FF in specific tissues, cells, and organs. Finally, we developed transgenic effector fish carrying the tetanus toxin light chain (TeTxLC) gene downstream of UAS, which is known to block synaptic transmission. We crossed the Gal4FF fish with the UAS:TeTxLC fish and analyzed double transgenic embryos for defects in touch response. From this analysis, we discovered that targeted expression of TeTxLC in distinct populations of neurons in the brain and the spinal cord caused distinct abnormalities in the touch response behavior. These studies illustrate that our Gal4FF gene trap and enhancer trap methods should be an important resource for genetic analysis of neuronal functions and behavior in vertebrates.

  19. Slit/Robo-mediated axon guidance in Tribolium and Drosophila: divergent genetic programs build insect nervous systems.

    PubMed

    Evans, Timothy A; Bashaw, Greg J

    2012-03-01

    As the complexity of animal nervous systems has increased during evolution, developmental control of neuronal connectivity has become increasingly refined. How has functional diversification within related axon guidance molecules contributed to the evolution of nervous systems? To address this question, we explore the evolution of functional diversity within the Roundabout (Robo) family of axon guidance receptors. In Drosophila, Robo and Robo2 promote midline repulsion, while Robo2 and Robo3 specify the position of longitudinal axon pathways. The Robo family has expanded by gene duplication in insects; robo2 and robo3 exist as distinct genes only within dipterans, while other insects, like the flour beetle Tribolium castaneum, retain an ancestral robo2/3 gene. Both Robos from Tribolium can mediate midline repulsion in Drosophila, but unlike the fly Robos cannot be down-regulated by Commissureless. The overall architecture and arrangement of longitudinal pathways are remarkably conserved in Tribolium, despite it having only two Robos. Loss of TcSlit causes midline collapse of axons in the beetle, a phenotype recapitulated by simultaneous knockdown of both Robos. Single gene knockdowns reveal that beetle Robos have specialized axon guidance functions: TcRobo is dedicated to midline repulsion, while TcRobo2/3 also regulates longitudinal pathway formation. TcRobo2/3 knockdown reproduces aspects of both Drosophila robo2 and robo3 mutants, suggesting that TcRobo2/3 has two functions that in Drosophila are divided between Robo2 and Robo3. The ability of Tribolium to organize longitudinal axons into three discrete medial-lateral zones with only two Robo receptors demonstrates that beetle and fly achieve equivalent developmental outcomes using divergent genetic programs.

  20. Genetic transformation of carnation (Dianthus caryophylus L.).

    PubMed

    Nontaswatsri, Chalermsri; Fukai, Seiichi

    2010-01-01

    This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

  1. Expression and production of cardiac angiogenic mediators depend on the Trypanosoma cruzi-genetic population in experimental C57BL/6 mice infection.

    PubMed

    Shrestha, Deena; Bajracharya, Bijay; Paula-Costa, Guilherme; Salles, Beatriz C; Leite, Ana Luísa J; Menezes, Ana Paula J; Souza, Débora Ms; Oliveira, Laser Am; Talvani, André

    2017-03-01

    Mammalian cardiac cells are important targets to the protozoan Trypanosoma cruzi. The inflammatory reaction in the host aims at eliminating this parasite, can lead to cell destruction, fibrosis and hypoxia. Local hypoxia is well-defined stimulus to the production of angiogenesis mediators. Assuming that different genetic T. cruzi populations induce distinct inflammation and disease patterns, the current study aims to investigate whether the production of inflammatory and angiogenic mediators is a parasite strain-dependent condition. C57BL/6 mice were infected with the Y and Colombian strains of T. cruzi and euthanized at the 12th and 32nd days, respectively. The blood and heart tissue were processed in immune assays and/or qPCR (TNF, IL-17, IL-10, CCL2, CCL3, CCL5, CCR2, CCR5 and angiogenic factors VEGF, Ang-1, Ang-2) and in histological assays. The T. cruzi increased the inflammatory and angiogenic mediators in the infected mice when they were compared to non-infected animals. However, the Colombian strain has led to higher (i) leukocyte infiltration, (ii) cardiac TNF and CCL5 production/expression, (iii) cardiac tissue parasitism, and to higher (iv) ratio between heart/body weights. On the other hand, the Colombian strain has caused lower production and expression VEGF, Ang-1 and Ang-2, when it was compared to the Y strain of the parasite. The present study highlights that the T. cruzi-genetic population defines the pattern of angiogenic/inflammatory mediators in the heart tissue, and that it may contribute to the magnitude of the cardiac pathogenesis. Besides, such assumption opens windows to the understanding of the angiogenic mediator's role in association with the experimental T. cruzi infection.

  2. Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter

    PubMed Central

    White, G. F.; Snape, J. R.; Nicklin, S.

    1996-01-01

    Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen. PMID:16535244

  3. Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.

    PubMed

    Akutsu, M; Ishizaki, T; Sato, H

    2004-03-01

    An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.

  4. Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells 1

    PubMed Central

    Neff, Nicola T.; Binns, Andrew N.

    1985-01-01

    Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis. Images Fig. 2 PMID:16664024

  5. A Genetic Screen Identifies a Requirement for Cysteine-Rich–Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity

    PubMed Central

    Chern, Mawsheng; Xu, Qiufang; Bart, Rebecca S.; Bai, Wei; Ruan, Deling; Sze-To, Wing Hoi; Canlas, Patrick E.; Jain, Rashmi; Chen, Xuewei; Ronald, Pamela C.

    2016-01-01

    Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression. PMID:27176732

  6. Tryptophan kynurenine metabolism as a common mediator of genetic and environmental impacts in major depressive disorder: the serotonin hypothesis revisited 40 years later.

    PubMed

    Oxenkrug, Gregory F

    2010-01-01

    The original 1969 Lancet paper proposed in depression the activity of liver tryptophan-pyrrolase is stimulated by raised blood corticosteroids levels, and metabolism of tryptophan is shunted away from serotonin production, and towards kynurenine production. Discovery of neurotropic activity of kynurenines suggested that up-regulation of the tryptophan-kynurenine pathway not only augmented serotonin deficiency but also underlined depression-associated anxiety, psychosis and cognitive decline. The present review of genetic and hormonal factors regulating kynurenine pathway of tryptophan metabolism suggests that this pathway mediates both genetic and environmental mechanisms of depression. Rate-limiting enzymes of kynurenine formation, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are activated by stress hormones (TDO) and/or by pro-inflammatory cytokines (IDO). Simultaneous presence of high producers alleles of proinflammatory cytokines genes (e.g., interferon-gamma and tumor necrosis factor-alpha) determines the genetic predisposition to depression via up-regulation of IDO while impact of environmental stresses is mediated via hormonal activation of TDO. Tryptophan-kynurenine pathway represents a major meeting point of gene-environment interaction in depression and a new target for pharmacological intervention.

  7. Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.).

    PubMed

    Zhang, Zhifen; Finer, John J

    2016-01-01

    Agrobacterium-mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 10(8)-10(9) bacteria mL(-1), followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 10(2) bacteria mL(-1)) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 10(8) bacteria mL(-1)) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

  8. Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules.

    PubMed

    Chen, Liang; Cong, Yuanyuan; He, Hongxia; Yu, Ying

    2014-02-10

    A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2-3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly.

  9. Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum.

    PubMed

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Jones, Jeffrey B; Obradović, Aleksa

    2015-09-01

    Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (<92%) between the whole genome sequences of strain KFB 330(T) and related Agrobacterium species. The major cellular fatty acids of the novel strains were 18:1 w7c (72.8-77.87%) and 16:0 (6.82-8.58%). Phenotypic features allowed their differentiation from closely related species. Polyphasic characterization showed that the three strains represent a novel species of the genus Agrobacterium, for which the name Agrobacterium arsenijevicii sp. nov. is proposed. The type strain of A. arsenijevicii is KFB 330(T) (= CFBP 8308(T) = LMG 28674(T)).

  10. Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum.

    PubMed

    Ooi, Chai Theam; Syahida, Ahmad; Stanslas, Johnson; Maziah, Mahmood

    2013-03-01

    This article presents the abilities and efficiencies of five different strains of Agrobacterium rhizogenes (strain ATCC 31798, ATCC 43057, AR12, A4 and A13) to induce hairy roots on Solanum mammosum through genetic transformation. There is significant difference in the transformation efficiency (average number of days of hairy root induction) and transformation frequency for all strains of A. rhizogenes (P < 0.05). Both A. rhizogenes strain AR12 and A13 were able to induce hairy root at 6 days of co-cultivation, which were the fastest among those tested. However, the transformation frequencies of all five strains were below 30 %, with A. rhizogenes strain A4 and A13 showing the highest, which were 21.41 ± 10.60 % and 21.43 ± 8.13 % respectively. Subsequently, the cultures for five different hairy root lines generated by five different strains of bacteria were established. However, different hairy root lines showed different growth index under the same culture condition, with the hairy root lines induced by A. rhizogenes strain ATCC 31798 exhibited largest increase in fresh biomass at 45 days of culture under 16 h light/8 h dark photoperiod in half-strength MS medium. The slowest growing hairy root line, which was previously induced by A. rhizogenes strain A13, when cultured in optimized half-strength MS medium containing 1.5 times the standard amount of ammonium nitrate and potassium nitrate and 5 % (w/v) sucrose, had exhibited improvement in growth index, that is, the fresh biomass was almost double as compared to its initial growth in unmodified half-strength MS medium.

  11. Characterization of the mmsAB-araD1 (gguABC) genes of Agrobacterium tumefaciens.

    PubMed

    Zhao, Jinlei; Binns, Andrew N

    2011-12-01

    The chvE-gguABC operon plays a critical role in both virulence and sugar utilization through the activities of the periplasmic ChvE protein, which binds to a variety of sugars. The roles of the GguA, GguB, and GguC are not known. While GguA and GguB are homologous to bacterial ABC transporters, earlier genetic analysis indicated that they were not necessary for utilization of sugars as the sole carbon source. To further examine this issue, in-frame deletions were constructed separately for each of the three genes. Our growth analysis clearly indicated that GguA and GguB play a role in sugar utilization and strongly suggests that GguAB constitute an ABC transporter with a wide range of substrates, including L-arabinose, D-fucose, D-galactose, D-glucose, and D-xylose. Site-directed mutagenesis showed that a Walker A motif was vital to the function of GguA. We therefore propose renaming gguAB as mmsAB, for multiple monosaccharide transport. A gguC deletion affected growth only on L-arabinose medium, suggesting that gguC encodes an enzyme specific to L-arabinose metabolism, and this gene was renamed araD1. Results from bioinformatics and experimental analyses indicate that Agrobacterium tumefaciens uses a pathway involving nonphosphorylated intermediates to catabolize L-arabinose via an L-arabinose dehydrogenase, AraA(At), encoded at the Atu1113 locus.

  12. Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus.

    PubMed

    Alam, Pravej; Khan, Zainul Abdeen; Abdin, Malik Zainul; Khan, Jawaid A; Ahmad, Parvaiz; Elkholy, Shereen F; Sharaf-Eldin, Mahmoud A

    2017-05-01

    Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L(-1) (BAP) and 0.5 mg L(-1) (NAA) while 80% shoot percentage obtained with 4.0 mg L(-1) (BAP) and 0.05 mg L(-1) (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.

  13. Effective elimination of chimeric tissue in transgenics for the stable genetic transformation of lesquerella fendleri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 carrying binary vector pCAMBIA 1301.1...

  14. Topological characterization of an inner membrane (1-->3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749.

    PubMed

    Karnezis, Tara; Epa, V Chandana; Stone, Bruce A; Stanisich, Vilma A

    2003-10-01

    The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of beta-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31-41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of approximately 60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of approximately 420 kDa and approximately 500 kDa.

  15. Examining the Use of Distance-Mediated Case Conferencing for Case-Based Training in Clinical Cancer Genetics

    ERIC Educational Resources Information Center

    Blazer, Kathleen R.

    2010-01-01

    Individuals with a cancer-predisposing genetic trait have a lifetime risk to develop cancer approaching 100 percent, and cancer often strikes early in age, before standard recommended cancer screening begins. Identifying hereditary cancer predisposition through genetic cancer risk assessment (GCRA) allows for intensified measures to prevent…

  16. Draft Genome Sequence of Agrobacterium sp. Strain R89-1, a Morphine Alkaloid-Biotransforming Bacterium

    PubMed Central

    Kyslíková, Eva

    2016-01-01

    Agrobacterium sp. strain R89-1 isolated from composted wastes of Papaver somniferum can effectively biotransform codeine/morphine into 14-OH-derivatives. Here, we present a 4.7-Mb assembly of the R89-1 strain genome. The draft shows that the strain R89-1 represents a distinct phylogenetic lineage within the genus Agrobacterium. PMID:27056219

  17. Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it difficult to obtain transgenic plants?

    PubMed

    Chovelon, V; Restier, V; Giovinazzo, N; Dogimont, C; Aarrouf, J

    2011-11-01

    Melon (Cucumis melo L.) is widely considered as a recalcitrant species for genetic transformation. In this study, we developed different regeneration and transformation protocols and we examined the regeneration process at different steps by histological studies. The highest regeneration rate (1.13 ± 0.02 plants per explant) was obtained using cotyledon explants of the 'Védrantais' genotype on Murashige and Skoog (MS) medium supplemented with 0.2 mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l dimethylallylaminopurine (2-iP). Agrobacterium tumefaciens-mediated transformations with the uidA reporter gene were realized on cotyledon explants cultivated in these conditions: 70-90% of explants expressed a transient GUS activity during the early stages of regeneration, however, only few transgenic plants were obtained (1.8-4.5% of stable transformation with the GV2260pBI101 strain). These results revealed a low capacity of melon GUS-positive cells to regenerate transgenic plants. To evaluate the influence of the Agrobacterium infection on plant regeneration, histological analyses were conducted on explants 2, 7, 15, and 28 days after co-culture with the GV2260pBI101 strain. Genetic transformation occurred in epidermal and sub-epidermal cells and reached the meristematic structures expressing a high level of GUS activity during 14 days of culture; but after this period, most of the meristematic structures showed premature cell vacuolization and disorganization. This disruption of the GUS-positive meristematic areas could be responsible of the difficulties encountered to regenerate melon plants after genetic transformation.

  18. [Construction of a bivalent plant expression vector carrying VvSUC11 and VvSUC12 genes and its genetic transformation in sugar beet].

    PubMed

    Yin, Donglin; Zhu, Jianbo; Wang, Aiying; Xiang, Benchun

    2011-08-01

    We have recombined genes VvSUC11, VvSUC12 from Vitis vinifera L., and root-specific promoters of sweet potato storage protein gene from Ipomoea batatas L. Lam., named as SP1 and SP2. We have constructed a vector pCAMBIA2301-SP1- VvSUC11-SP2-VvSUC12 using pCAMBIA2301 as an original vector. VvSUC11 and VvSUC12 were under the control of root-specific promoters of sweet potato storage protein gene. We transformed the vector into KWS-9103 breeding line of Beta vulgaris L. with Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as following: the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD(600)=0.5, supplemented with 0.005% Silwet L-77, and followed by a 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin-resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the target genes had integrated into sugar beet genome and expressed. It will lay a foundation for further studying their function in Beta vulgaris.

  19. Production of insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140).

    PubMed

    Portilho, Márcia; Matioli, Graciette; Zanin, Gisella Maria; de Moraes, Flávio Faria; Scamparini, Adilma Regina Pippa

    2006-01-01

    Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).

  20. Production of insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140).

    PubMed

    Portilho, Márcia; Matioli, Graciette; Zanin, Gisella Maria; de Moraes, Flávio Faria; Scamparini, Adilma Regina Pippa

    2006-03-01

    Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).

  1. Range expansion of house sparrows (Passer domesticus) in Kenya: evidence of genetic admixture and human-mediated dispersal.

    PubMed

    Schrey, Aaron W; Liebl, Andrea L; Richards, Christina L; Martin, Lynn B

    2014-01-01

    Introduced species offer an opportunity to study the ecological process of range expansions. Recently, 3 mechanisms have been identified that may resolve the genetic paradox (the seemingly unlikely success of introduced species given the expected reduction in genetic diversity through bottlenecks or founder effects): multiple introductions, high propagule pressure, and epigenetics. These mechanisms are probably also important in range expansions (either natural or anthropogenic), yet this possibility remains untested in vertebrates. We used microsatellite variation (7 loci) in house sparrows (Passer domesticus), an introduced species that has been spreading across Kenya for ~60 years, to determine if patterns of variation could explain how this human commensal overcame the genetic paradox and expresses such considerable phenotypic differentiation across this new range. We note that in some cases, polygenic traits and epistasis among genes, for example, may not have negative effects on populations. House sparrows arrived in Kenya by a single introduction event (to Mombasa, ~1950) and have lower genetic diversity than native European and introduced North American populations. We used Bayesian clustering of individuals (n = 233) to detect that at least 2 types of range expansion occurred in Kenya: one with genetic admixture and one with little to no admixture. We also found that genetic diversity increased toward a range edge, and the range expansion was consistent with long-distance dispersal. Based on these data, we expect that the Kenyan range expansion was anthropogenically influenced, as the expansions of other introduced human commensals may also be.

  2. Influence of long-distance seed dispersal on the genetic diversity of seed rain in fragmented Pinus densiflora populations relative to pollen-mediated gene flow.

    PubMed

    Ozawa, Hajime; Watanabe, Atsushi; Uchiyama, Kentaro; Saito, Yoko; Ide, Yuji

    2013-01-01

    Long-distance dispersal (LDD) of seeds has a critical impact on species survival in patchy landscapes. However, relative to pollen dispersal, empirical data on how seed LDD affects genetic diversity in fragmented populations have been poorly reported. Thus, we attempted to indirectly evaluate the influence of seed LDD by estimating maternal and paternal inbreeding in the seed rain of fragmented 8 Pinus densiflora populations. In total, the sample size was 458 seeds and 306 adult trees. Inbreeding was estimated by common parentage analysis to evaluate gene flow within populations and by sibship reconstruction analysis to estimate gene flow within and among populations. In the parentage analysis, the observed probability that sampled seeds had the same parents within populations was significantly larger than the expected probability in many populations. This result suggested that gene dispersal was limited to within populations. In the sibship reconstruction, many donors both within and among populations appeared to contribute to sampled seeds. Significant differences in sibling ratios were not detected between paternity and maternity. These results suggested that seed-mediated gene flow and pollen-mediated gene flow from outside population contributed some extent to high genetic diversity of the seed rain (H E > 0.854). We emphasize that pine seeds may have excellent potential for gene exchange within and among populations.

  3. Consequences of frugivore-mediated seed dispersal for the spatial and genetic structures of a neotropical palm.

    PubMed

    Choo, Juanita; Juenger, T E; Simpson, B B

    2012-02-01

    The idiosyncratic behaviours of seed dispersers are important contributors to plant spatial associations and genetic structures. In this study, we used a combination of field, molecular and spatial studies to examine the connections between seed dispersal and the spatial and genetic structures of a dominant neotropical palm Attalea phalerata. Field observation and genetic parentage analysis both indicated that the majority of A. phalerata seeds were dispersed locally over short distances (<30 m from the maternal tree). Spatial and genetic structures between adults and seedlings were consistent with localized and short-distance seed dispersal. Dispersal contributed to spatial associations among maternal sibling seedlings and strong spatial and genetic structures in both seedlings dispersed near (<10 m) and away (>10 m) from maternal palms. Seedlings were also spatially aggregated with juveniles. These patterns are probably associated with the dispersal of seeds by rodents and the survival of recruits at specific microsites or neighbourhoods over successive fruiting periods. Our cross-cohort analyses found palms in older cohorts and cohort pairs were associated with a lower proportion of offspring and sibling neighbours and exhibited weaker spatial and genetic structures. Such patterns are consistent with increased distance- and density-dependent mortality over time among palms dispersed near maternal palms or siblings. The integrative approaches used for this study allowed us to infer the importance of seed dispersal activities in maintaining the aggregated distribution and significant genetic structures among A. phalerata palms. We further conclude that distance- and density-dependent mortality is a key postdispersal process regulating this palm population.

  4. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    PubMed

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

  5. Sulcal depth-position profile is a genetically mediated neuroscientific trait: description and characterization in the central sulcus.

    PubMed

    McKay, D Reese; Kochunov, Peter; Cykowski, Matthew D; Kent, Jack W; Laird, Angela R; Lancaster, Jack L; Blangero, John; Glahn, David C; Fox, Peter T

    2013-09-25

    Genetic and environmental influences on brain morphology were assessed in an extended-pedigree design by extracting depth-position profiles (DPP) of the central sulcus (CS). T1-weighted magnetic resonance images were used to measure CS length and depth in 467 human subjects from 35 extended families. Three primary forms of DPPs were observed. The most prevalent form, present in 70% of subjects, was bimodal, with peaks near hand and mouth regions. Trimodal and unimodal configurations accounted for 15 and 8%, respectively. Genetic control accounted for 56 and 66% of between-subject variance in average CS depth and length, respectively, and was not significantly influenced by environmental factors. Genetic control over CS depth ranged from 1 to 50% across the DPP. Areas of peak heritability occurred at locations corresponding to hand and mouth areas. Left and right analogous CS depth measurements were strongly pleiotropic. Shared genetic influence lessened as the distance between depth measurements was increased. We argue that DPPs are powerful phenotypes that should inform genetic influence of more complex brain regions and contribute to gene discovery efforts.

  6. Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

    To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed

  7. Psychosocial mechanisms of serotonin transporter's genetic polymorphism in susceptibility to major depressive disorder: mediated by trait coping styles and interacted with life events.

    PubMed

    Wang, Yanfang; Sun, Ning; Liu, Zhifen; Li, Xinrong; Yang, Chunxia; Zhang, Kerang

    2016-01-01

    The mechanism of 5-HTT genetic polymorphisms related susceptibility of major depressive disorder (MDD) has not been fully understood. Two hundred MDD patients and 199 control subjects were included. A model of two binary logistical regressions with and without controlling for different psychosocial variables, was applied to investigate the possible mediation effects of psychosocial factors in contribution of 5-HTT polymorphisms in MDD development. These psychosocial variables included personality, trait coping style, life events and social support. Then, contribution of interactions between 5-HTT polymorphisms and psychosocial factors in MDD was investigated by a stepwise logistical approach. The results indicated a significant association of 5-HTT LPR with the MDD indicence, but not of VNTR genotype variances with the MDD incidence without counting effects of psychosocial factors. The ss genotype of LPR demonstrated 2.50 (95% CI: 1.11-5.62) times higher odds to develop MDD than ll genotype (p=0.026). After including psychosocial variables, the odds ratio of 5-HTT LPR ss to ll genotype dropped to 1.30 times (95% CI: 0.41-4.10) and became non-significant (p=0.658). While psychosocial variables all showed significant contributions to MDD susceptibility. Our data suggested an intermediator role of this psychosocial variable in LPR genetic pathogenesis of MDD. Whereas, 5-HTT VNTR could significantly affect MDD outcome by interacting with life events (p=0.043). In conclusion, 5-HTT LPR and VNTR polymorphisms could affect MDD susceptibility through mediation by trait coping styles and interaction with severe life events, respectively. The genetic information of 5-HTT can be potentially helpful for diagnosis and further therapy.

  8. Psychosocial mechanisms of serotonin transporter’s genetic polymorphism in susceptibility to major depressive disorder: mediated by trait coping styles and interacted with life events

    PubMed Central

    Wang, Yanfang; Sun, Ning; Liu, Zhifen; Li, Xinrong; Yang, Chunxia; Zhang, Kerang

    2016-01-01

    The mechanism of 5-HTT genetic polymorphisms related susceptibility of major depressive disorder (MDD) has not been fully understood. Two hundred MDD patients and 199 control subjects were included. A model of two binary logistical regressions with and without controlling for different psychosocial variables, was applied to investigate the possible mediation effects of psychosocial factors in contribution of 5-HTT polymorphisms in MDD development. These psychosocial variables included personality, trait coping style, life events and social support. Then, contribution of interactions between 5-HTT polymorphisms and psychosocial factors in MDD was investigated by a stepwise logistical approach. The results indicated a significant association of 5-HTT LPR with the MDD indicence, but not of VNTR genotype variances with the MDD incidence without counting effects of psychosocial factors. The ss genotype of LPR demonstrated 2.50 (95% CI: 1.11-5.62) times higher odds to develop MDD than ll genotype (p=0.026). After including psychosocial variables, the odds ratio of 5-HTT LPR ss to ll genotype dropped to 1.30 times (95% CI: 0.41-4.10) and became non-significant (p=0.658). While psychosocial variables all showed significant contributions to MDD susceptibility. Our data suggested an intermediator role of this psychosocial variable in LPR genetic pathogenesis of MDD. Whereas, 5-HTT VNTR could significantly affect MDD outcome by interacting with life events (p=0.043). In conclusion, 5-HTT LPR and VNTR polymorphisms could affect MDD susceptibility through mediation by trait coping styles and interaction with severe life events, respectively. The genetic information of 5-HTT can be potentially helpful for diagnosis and further therapy. PMID:27158415

  9. Social Structure and Genetic Distance Mediate Nestmate Recognition and Aggressiveness in the Facultative Polygynous Ant Pheidole pallidula

    PubMed Central

    De Laet, Sophie; Lenoir, Alain; Passera, Luc; Aron, Serge

    2016-01-01

    In social insects, the evolutionary stability of cooperation depends on the privileged relationships between individuals of the social group, which is facilitated by the recognition of relatives. Nestmate recognition is based on genetically determined cues and/or environmentally derived chemical components present on the cuticle of individuals. Here, we studied nestmate recognition in the ant Pheidole pallidula, a species where both single-queen (monogyne) and multiple-queen (polygyne) colonies co-occur in the same population. We combined geographical, genetic and chemical analyses to disentangle the factors influencing the level of intraspecific aggressiveness. We show that encounters between workers from neighbouring colonies (i.e., nests less than 5 m away) are on average less aggressive than those between workers from more distant colonies. Aggressive behaviour is associated with the level of genetic difference: workers from monogyne colonies are more aggressive than workers from polygyne colonies, and the intensity of aggressiveness is positively associated with the genetic distance between colonies. Since the genetic distance is correlated with the spatial distance between pairs of colonies, the lower level of aggression toward neighbours may result from their higher relatedness. In contrast, the analysis of overall cuticular hydrocarbon profiles shows that aggressive behaviour is associated neither with the chemical diversity of colonies, nor with the chemical distances between them. When considering methyl-branched alkanes only, however, chemical distances differed between monogyne and polygyne colonies and were significantly associated with aggressiveness. Altogether, these results show that the social structure of colonies and the genetic distances between colonies are two major factors influencing the intensity of agonistic behaviours in the ant P. pallidula. PMID:27243627

  10. Genetic variability of aryl hydrocarbon receptor (AhR)-mediated regulation of the human UDP glucuronosyltransferase (UGT) 1A4 gene

    SciTech Connect

    Erichsen, Thomas J.; Ehmer, Ursula; Kalthoff, Sandra; Lankisch, Tim O.; Mueller, Tordis M.; Munzel, Peter A.; Manns, Michael P.; Strassburg, Christian P.

    2008-07-15

    UDP glucuronosyltransferases (UGTs) play an important role for drug detoxification and toxicity. UGT function is genetically modulated by single nucleotide polymorphisms (SNPs) which lead to the expression of functionally altered protein, or altered expression levels. UGT1A4 activity includes anticonvulsants, antidepressants and environmental mutagens. In this study the induction of the human UGT1A4 gene and a potential influence of genetic variation in its promoter region were analyzed. SNPs at bp - 219 and - 163 occurred in 9% among 109 blood donors reducing UGT1A4 transcription by 40%. UGT1A4 transcription was dioxin inducible. Reporter gene experiments identified 2 xenobiotic response elements (XRE), which were functionally confirmed by mutagenesis analyses, and binding was demonstrated by electromobility shift assays. Constitutive human UGT1A4 gene expression and induction was aryl hydrocarbon receptor (AhR)-dependent, and reduced in the presence of SNPs at bp - 219 and - 163. AhR-mediated regulation of the human UGT1A4 gene by two XRE and a modulation by naturally occurring genetic variability by SNPs is demonstrated, which indicates gene-environment interaction with potential relevance for drug metabolism.

  11. Genome Sequence of Agrobacterium tumefaciens Strain F2, a Bioflocculant-Producing Bacterium

    PubMed Central

    Li, Ang; Geng, Jianing; Cui, Di; Shu, Chang; Zhang, Si; Yang, Jixian; Xing, Jie; Wang, Jinna; Ma, Fang; Hu, Songnian

    2011-01-01

    Agrobacterium tumefaciens F2 is an efficient bioflocculant-producing bacterium. But the genes related to the metabolic pathway of bioflocculant biosynthesis in strain F2 are unknown. We present the draft genome of A. tumefaciens F2. It could provide further insight into the biosynthetic mechanism of polysaccharide-like bioflocculant in strain F2. PMID:21914861

  12. Genome sequence of Agrobacterium tumefaciens strain F2, a bioflocculant-producing bacterium.

    PubMed

    Li, Ang; Geng, Jianing; Cui, Di; Shu, Chang; Zhang, Si; Yang, Jixian; Xing, Jie; Wang, Jinna; Ma, Fang; Hu, Songnian

    2011-10-01

    Agrobacterium tumefaciens F2 is an efficient bioflocculant-producing bacterium. But the genes related to the metabolic pathway of bioflocculant biosynthesis in strain F2 are unknown. We present the draft genome of A. tumefaciens F2. It could provide further insight into the biosynthetic mechanism of polysaccharide-like bioflocculant in strain F2.

  13. An enrichment technique for auxotrophs of Agrobacterium tumefaciens using a combination of carbenicillin and lysozyme.

    PubMed

    Klapwijk, P M; de Jonge, A J; Schilperoort, R A; Rörsch, A

    1975-11-01

    A procedure to enrich for auxotrophic and fermentation mutants of Agrobacterium tumefaciens is described. The method is based on the amplification of the killing power of carbenicillin by the addition of lysozyme. Isolation frequencies of some types of mutants are presented, with and without the application of the proposed procedure. The yield of mutants is usually enhanced a hundredfold per enrichment treatment.

  14. Nodulation of Sesbania Species by Rhizobium (Agrobacterium) Strain IRBG74 and Other Rhizobia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridisation with R. ...

  15. 77 FR 40880 - Agrobacterium radiobacter; Registration Review Proposed Decision; Notice of Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-11

    ...: Ann Sibold, Regulatory Action Leader, Biopesticides and Pollution Prevention Division (7511P), Office... occurring soil bacterium present in many soil types. Two strains of Agrobacterium radiobacter, K84/Kerr-84.... Matthews, Director, Biopesticides and Pollution Prevention Division, Office of Pesticide Programs....

  16. The Limits of Child Effects: Evidence for Genetically Mediated Child Effects on Corporal Punishment but Not on Physical Maltreatment

    ERIC Educational Resources Information Center

    Jaffee, Sara R.; Caspi, Avshalom; Moffitt, Terrie E.; Polo-Tomas, Monica; Price, Thomas S.; Taylor, Alan

    2004-01-01

    Research on child effects has demonstrated that children's difficult and coercive behavior provokes harsh discipline from adults. Using a genetically sensitive design, the authors tested the limits of child effects on adult behavior that ranged from the normative (corporal punishment) to the nonnormative (physical maltreatment). The sample was a…

  17. LRRK2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a Drosophila model: suppression by curcumin.

    PubMed

    Yang, Dejun; Li, Tianxia; Liu, Zhaohui; Arbez, Nicolas; Yan, Jianqun; Moran, Timothy H; Ross, Christopher A; Smith, Wanli W

    2012-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.

  18. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.

    PubMed

    Wu, Yuxuan; Zhou, Hai; Fan, Xiaoying; Zhang, Ying; Zhang, Man; Wang, Yinghua; Xie, Zhenfei; Bai, Meizhu; Yin, Qi; Liang, Dan; Tang, Wei; Liao, Jiaoyang; Zhou, Chikai; Liu, Wujuan; Zhu, Ping; Guo, Hongshan; Pan, Hong; Wu, Chunlian; Shi, Huijuan; Wu, Ligang; Tang, Fuchou; Li, Jinsong

    2015-01-01

    Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

  19. Complex Craniofacial Changes in Blind Cave-Dwelling Fish Are Mediated by Genetically Symmetric and Asymmetric Loci

    PubMed Central

    Gross, Joshua B.; Krutzler, Amanda J.; Carlson, Brian M.

    2014-01-01

    The genetic regulators of regressive craniofacial morphologies are poorly understood. To shed light on this problem, we examined the freshwater fish Astyanax mexicanus, a species with surface-dwelling and multiple independent eyeless cave-dwelling forms. Changes affecting the skull in cavefish include morphological alterations to the intramembranous circumorbital bones encircling the eye. Many of these modifications, however, have evolved separately from eye loss, such as fragmentation of the third suborbital bone. To understand the genetic architecture of these eye-independent craniofacial alterations, we developed and scored 33 phenotypes in the context of an F2 hybrid mapping pedigree bred from Pachón cavefish and surface fish. We discovered several individuals exhibiting dramatic left–right differences in bone formation, such as extensive fragmentation on the right side only. This observation, along with well-known eye size asymmetry in natural cave-dwelling animals, led us to further evaluate left–right genetic differences for the craniofacial complex. We discovered three phenotypes, inclusive of bone fragmentation and fusion, which demonstrated a directional heritable basis only on one side. Interestingly, the overall areas of affected bones were genetically symmetric. Phenotypic effect plots of these novel craniofacial QTL revealed that cave alleles are associated with abnormal conditions such as bony fusion and fragmentation. Moreover, many linked loci overlapped with other cave-associated traits, suggesting regressive craniofacial changes may evolve through linkage or as antagonistic pleiotropic consequences of cave-associated adaptations. These novel findings illuminate significant craniofacial changes accompanying evolution in complete darkness and reveal complex changes to the skull differentially influenced by genetic changes affecting the left and right sides. PMID:24496009

  20. SS1 (NAL1)- and SS2-Mediated Genetic Networks Underlying Source-Sink and Yield Traits in Rice (Oryza sativa L.).

    PubMed

    Xu, Jian-Long; Wang, Yun; Zhang, Fan; Wu, Yuan; Zheng, Tian-Qing; Wang, Yong-Hong; Zhao, Xiu-Qin; Cui, Yan-Ru; Chen, Kai; Zhang, Qiang; Lin, Hong-Xuan; Li, Jia-Yang; Li, Zhi-Kang

    2015-01-01

    Source leaf/sink capacity (SS) traits are important determinants of grain yield (GY) of rice. To understand the genetic basis of the SS relationship in rice, five SS and GY traits of rice were genetically dissected using two reciprocal introgression populations. Seventy-three QTL affecting the SS and GY traits were identified, most of which were detected in one of the parental genetic backgrounds (GBs). Two major QTL at bins 4.7 (SS1) and 3.12 (SS2) were associated consistently with all measured SS and yield traits in both GBs across two contrasting environments. Strong interactions between SS1/SS2 and the detected QTL led us to the discovery of genetic networks affecting the SS and GY traits. The SS1 acted as a regulator controlling two groups of downstream QTL affecting the source leaf width and grain number per panicle (GNP). SS2 functioned as a regulator positively regulating different groups of downstream QTL affecting the source leaf length, GNP, grain weight, and GY. Map-based cloning of SS1 indicates that SS1 is NAL1 involved in polar auxin/IAA transport. Different alleles at NAL1 were apparently able to qualitatively and/or quantitatively control the IAA transport from the apical meristem to different plant tissues and thus regulate those downstream loci/pathways controlling different SS traits of rice. There was a functional allele and a non-functional mutation in the parents at each of the QTL downstream of SS1 or SS2, which were detectable only in the presence of the functional allele of SS1 or SS2. Our results provided direct evidence that SS and yield traits in rice are controlled by complex signaling pathways and suggest further improvement of rice yield potential with enhanced and balanced SS relationships can be achieved by accurately manipulating allelic combinations at loci in the SS1 and SS2 mediated pathways.

  1. Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phomopsis seed decay (PSD) of soybean is caused primarily by the fungal pathogen Phomopsis longicolla. PSD impairs seed germination, reduces seedling vigor, and can substantially reduce stand establishment. In hot and humid conditions, PSD can cause significant yield losses. Few studies have explore...

  2. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    PubMed

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions.

  3. Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens

    PubMed Central

    FAN, ZHONGQI; YU, HUIMEI; GUO, QI; HE, DAN; XUE, BAIJI; XIE, XIANGLI; YOKOYAMA, KOJI; WANG, LI

    2016-01-01

    Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well-organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti-oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly-inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced-polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix-loop-helix transcription factor coding region. A decrease in the levels of anti-oxidative stress-associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress. PMID:26847000

  4. A central role of abscisic acid in drought stress protection of Agrobacterium-induced tumors on Arabidopsis.

    PubMed

    Efetova, Marina; Zeier, Jürgen; Riederer, Markus; Lee, Chil-Woo; Stingl, Nadja; Mueller, Martin; Hartung, Wolfram; Hedrich, Rainer; Deeken, Rosalia

    2007-11-01

    Crown gall tumors induced by Agrobacterium tumefaciens represent a sink that has to be provided with nutrients and water by the host plant. The lack of an intact epidermis or cuticle results in uncontrolled loss of water. However, neither the tumor nor the host plant displays wilting. This phenomenon points to drought adaptation in both tumors and the crown gall host plant. To understand the underlying molecular mechanisms of protection against desiccation the gene expression pattern of Arabidopsis (Arabidopsis thaliana) tumors was integrated with the profile of stress metabolites: Arabidopsis tumors accumulated high amounts of abscisic acid (ABA), the ethylene precursor aminocyclopropyl carboxylic acid, osmoprotectants, and form a suberized periderm-like protective layer. Suberization of the outer tumor cell layers most likely is mediated by ABA since external application of ABA induced suberization of Arabidopsis roots. However, the expression level of the classical marker genes, known to respond to drought stress and/or ABA, was lower in tumors. Instead another set of drought and/or ABA-inducible genes was more highly transcribed. Elevated transcription of several ABA-dependent aquaporin genes might indicate that ABA controls the water balance of the tumor. The retarded tumor growth on abi and aba mutant plants underlined the importance of a tumor-specific ABA signaling pathway. Taken together, we propose that ABA is an important signal for protection of tumors against desiccation and thus supports tumor development.

  5. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    PubMed

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

  6. AFLP genome scan in the black rat (Rattus rattus) from Madagascar: detecting genetic markers undergoing plague-mediated selection.

    PubMed

    Tollenaere, C; Duplantier, J-M; Rahalison, L; Ranjalahy, M; Brouat, C

    2011-03-01

    The black rat (Rattus rattus) is the main reservoir of plague (Yersinia pestis infection) in Madagascar's rural zones. Black rats are highly resistant to plague within the plague focus (central highland), whereas they are susceptible where the disease is absent (low altitude zone). To better understand plague wildlife circulation and host evolution in response to a highly virulent pathogen, we attempted to determine genetic markers associated with plague resistance in this species. To this purpose, we combined a population genomics approach and an association study, both performed on 249 AFLP markers, in Malagasy R. rattus. Simulated distributions of genetic differentiation were compared to observed data in four independent pairs, each consisting of one population from the plague focus and one from the plague-free zone. We found 22 loci (9% of 249) with higher differentiation in at least two independent population pairs or with combining P-values over the four pairs significant. Among the 22 outlier loci, 16 presented significant association with plague zone (plague focus vs. plague-free zone). Population genetic structure inferred from outlier loci was structured by plague zone, whereas the neutral loci dataset revealed structure by geography (eastern vs. western populations). A phenotype association study revealed that two of the 22 loci were significantly associated with differentiation between dying and surviving rats following experimental plague challenge. The 22 outlier loci identified in this study may undergo plague selective pressure either directly or more probably indirectly due to hitchhiking with selected loci.

  7. Genetic inactivation of pleiotrophin but not midkine potentiates clonidine-induced alpha-2 adrenergic-mediated analgesia.

    PubMed

    Vicente-Rodríguez, Marta; Pérez-García, Carmen; Gramage, Esther; Herradón, Gonzalo

    2013-09-01

    Genetic deletion of the heparin-binding cytokines pleiotrophin (PTN) or midkine (MK) potentiates morphine-induced antinociceptive effects in animal models. Despite the known interactions between the opioid and noradrenergic systems in the control of pain, the possible roles of PTN and/or MK in analgesia induced by agonists of α2-adrenergic receptors remained to be studied. We have now tested the antinociceptive effects of the α2-adrenergic receptor agonist clonidine in female PTN genetically deficient (PTN-/-), MK genetically deficient (MK-/-) and wild type (WT+/+) mice. We did not find differences among genotypes in the hot-plate test, an assay in which supraspinal and spinal mechanisms contribute to nociceptive responses, suggesting that endogenous expression of PTN and MK is not key in the analgesia induced by clonidine in this test. In contrast, we found that clonidine-induced analgesia was significantly enhanced in PTN-/- mice compared to MK-/- and WT+/+ mice in the tail-immersion test. Interestingly, the α2-adrenergic antagonist yohimbine prevented clonidine-induced analgesia in the tail immersion test in all the 3 genotypes. The data suggest that the spinal antinociceptive effects caused by stimulation of α2-adrenoceptors are differentially regulated by endogenous expression of PTN.

  8. Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris

    PubMed Central

    Peláez, Pablo; Hernández-López, Alejandrina; Estrada-Navarrete, Georgina; Sanchez, Federico

    2017-01-01

    Agrobacterium rhizogenes is a pathogenic bacteria that causes hairy root disease by transferring bacterial DNA into the plant genome. It is an essential tool for industry and research due to its capacity to produce genetically modified roots and whole organisms. Here, we identified and characterized small RNAs generated from the transfer DNA (T-DNA) of A. rhizogenes in hairy roots of common bean (Phaseolus vulgaris). Distinct abundant A. rhizogenes T-DNA-derived small RNAs (ArT-sRNAs) belonging to several oncogenes were detected in hairy roots using high-throughput sequencing. The most abundant and diverse species of ArT-sRNAs were those of 21- and 22-nucleotides in length. Many T-DNA encoded genes constituted phasiRNA producing loci (PHAS loci). Interestingly, degradome analysis revealed that ArT-sRNAs potentially target genes of P. vulgaris. In addition, we detected low levels of ArT-sRNAs in the A. rhizogenes-induced calli generated at the wound site before hairy root emergence. These results suggest that RNA silencing targets several genes from T-DNA of A. rhizogenes in hairy roots of common bean. Therefore, the role of RNA silencing observed in this study has implications in our understanding and usage of this unique plant-bacteria interaction. PMID:28203245

  9. Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA

    PubMed Central

    Yamada, Tetsuji; Palm, Curtis J.; Brooks, Bob; Kosuge, Tsune

    1985-01-01

    We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-1 gene of the octopine-type plasmid pTiA6NC from Agrobacterium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA. Images PMID:16593610

  10. Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris.

    PubMed

    Peláez, Pablo; Hernández-López, Alejandrina; Estrada-Navarrete, Georgina; Sanchez, Federico

    2017-01-01

    Agrobacterium rhizogenes is a pathogenic bacteria that causes hairy root disease by transferring bacterial DNA into the plant genome. It is an essential tool for industry and research due to its capacity to produce genetically modified roots and whole organisms. Here, we identified and characterized small RNAs generated from the transfer DNA (T-DNA) of A. rhizogenes in hairy roots of common bean (Phaseolus vulgaris). Distinct abundant A. rhizogenes T-DNA-derived small RNAs (ArT-sRNAs) belonging to several oncogenes were detected in hairy roots using high-throughput sequencing. The most abundant and diverse species of ArT-sRNAs were those of 21- and 22-nucleotides in length. Many T-DNA encoded genes constituted phasiRNA producing loci (PHAS loci). Interestingly, degradome analysis revealed that ArT-sRNAs potentially target genes of P. vulgaris. In addition, we detected low levels of ArT-sRNAs in the A. rhizogenes-induced calli generated at the wound site before hairy root emergence. These results suggest that RNA silencing targets several genes from T-DNA of A. rhizogenes in hairy roots of common bean. Therefore, the role of RNA silencing observed in this study has implications in our understanding and usage of this unique plant-bacteria interaction.

  11. Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid

    PubMed Central

    1983-01-01

    We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix- ). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods. PMID:6885919

  12. Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.).

    PubMed

    Swain, Swasti S; Rout, Kedar K; Chand, Pradeep K

    2012-10-01

    Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T(L)-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T(R)-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, (1)H-NMR, and (13)C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species.

  13. Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants.

    PubMed

    Bettini, Priscilla P; Marvasi, Massimiliano; Fani, Fabiola; Lazzara, Luigi; Cosi, Elena; Melani, Lorenzo; Mauro, Maria Luisa

    2016-10-01

    Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis.

  14. Phenolic content in differentiated tissue cultures of untransformed and Agrobacterium-transformed roots of anise (Pimpinella anisum L.).

    PubMed

    Andarwulan, N; Shetty, K

    1999-04-01

    To investigate the role of differentiation of anise tissue cultures on total phenolic and anethole contents, benzylaminopurine- and thidiazuron-induced shoot cultures were generated from roots of the A-8 clonal line and its Agrobacterium rhizogenes-induced genetically transformed derivative JB-10. Embryogenic cultures were induced following 2,4-D treatment. Root cultures were multiplied on hormone-free medium. The effect of proline on differentiation and phenolic synthesis was also investigated. GC/MS studies indicate that anethole was not produced in root or other differentiated cultures. The predominant phenolic metabolite, however, was an anethole precursor, epoxypseudoisoeugenol-2-methylbutyrate (EPB). Total phenolics and EPB contents were highest in root cultures, which also correlated with higher proline content. Embryo and shoot cultures had reduced phenolic level and EPB and proline contents. Antioxidant activity in all differentiating cultures was high on day 60 compared to that on day 30, and there was no significant difference between differentiating tissues. This indicated that antioxidant protection might be linked not only to phenolics but to other nonphenolic metabolites as well.

  15. Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies.

    PubMed

    Brevet, J; Tempé, J

    1988-03-01

    Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (TL-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the TL-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.

  16. PLZF, a Tumor Suppressor Genetically Lost in Metastatic Castration Resistant Prostate Cancer, is a Mediator of Resistance to Androgen Deprivation Therapy

    PubMed Central

    Hsieh, Chen-Lin; Botta, Ginevra; Gao, Shuai; Li, Tiantian; Van Allen, Eliezer M.; Treacy, Daniel J.; Cai, Changmeng; He, Housheng Hansen; Sweeney, Christopher J.; Brown, Myles; Balk, Steven P.; Nelson, Peter S.

    2015-01-01

    Whole exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveal that 5~7% of tumors harbor promyelocytic zinc finger protein (PLZF) homozygous deletions. PLZF is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT and this acquired mechanism together with the finding of genetic loss in CRPC implicate PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype. PMID:25808865

  17. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

    PubMed Central

    Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

    2017-01-01

    Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D

  18. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

    PubMed

    Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

    2016-01-01

    Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D

  19. Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.

    PubMed

    Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

    2013-03-01

    KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.

  20. Traffic, asthma and genetics: combining international birth cohort data to examine genetics as a mediator of traffic-related air pollution's impact on childhood asthma.

    PubMed

    MacIntyre, Elaina A; Carlsten, Christopher; MacNutt, Meaghan; Fuertes, Elaine; Melén, Eric; Tiesler, Carla M T; Gehring, Ulrike; Krämer, Ursula; Klümper, Claudia; Kerkhof, Marjan; Chan-Yeung, Moira; Kozyrskyj, Anita L; Berdel, Dietrich; Bauer, Carl Peter; Herbarth, Olf; Bauer, Mario; Schaaf, Beate; Koletzko, Sibylle; Pershagen, Goran; Brunekreef, Bert; Heinrich, Joachim; Brauer, Michael

    2013-07-01

    Associations between traffic-related air pollution and incident childhood asthma can be strengthened by analysis of gene-environment interactions, but studies have typically been limited by lack of study power. We combined data from six birth cohorts on: asthma, eczema and allergic rhinitis to 7/8 years, and candidate genes. Individual-level assessment of traffic-related air pollution exposure was estimated using land use regression or dispersion modeling. A total of 11,760 children were included in the Traffic, Asthma and Genetics (TAG) Study; 6.3 % reported physician-diagnosed asthma at school-age, 16.0 % had asthma at anytime during childhood, 14.1 % had allergic rhinitis at school-age, 10.0 % had eczema at school-age and 33.1 % were sensitized to any allergen. For GSTP1 rs1138272, the prevalence of heterozygosity was 16 % (range amongst individual cohorts, 11-17 %) and homozygosity for the minor allele was 1 % (0-2 %). For GSTP1 rs1695, the prevalence of heterozygosity was 45 % (40-48 %) and homozygosity for the minor allele, 12 % (10-12 %). For TNF rs1800629, the prevalence of heterozygosity was 29 % (25-32 %) and homozygosity for the minor allele, 3 % (1-3 %). TAG comprises a rich database, the largest of its kind, for investigating the effect of genotype on the association between air pollution and childhood allergic disease.

  1. Interactions between the Bumblebee Bombus pascuorum and Red Clover (Trifolium pratense) Are Mediated by Plant Genetic Background

    PubMed Central

    Sands, Richard J.; Rowntree, Jennifer K.

    2016-01-01

    Wildflower mixes are often planted around field margins to provide forage for pollinators. Although seed for these mixtures is often wild-sourced, for species where agricultural cultivars are available, for example red clover (Trifolium pratense), cultivars can also be included. Previous evidence suggests that plant genetic background can have a strong influence on plant-arthropod interactions and therefore the provenance and genetic background of the plants included in wildflower mixes could impact plant-pollinator interactions. We tested the performance of five individual T. pratense cultivars against two commercially available wild-sourced T. pratense populations in terms of their ability to attract potential pollinator species (focusing on bumblebees) and their floral traits using greenhouse and garden experiments. The main bumblebee observed interacting with T. pratense was Bombus pascuorum and we found no difference in the absolute number of B. pascuorum visiting the cultivars or wild populations. However, we found variation among cultivars and between wild populations in their ability to attract bumblebees, which seems to be related to their relative investment in different floral traits. There was a positive relationship between biomass and number of inflorescences produced by the wild populations of T. pratense, which was not apparent for the cultivars. This suggests that artificial selection on the cultivars has changed the G-matrix of correlated traits. We show that agricultural cultivars of T. pratense can be as effective as wild populations at attracting pollinators such as bumblebees, but that the genetic background of both cultivars and wild populations can have a significant impact on the attractiveness of the plant to pollinators. We also show divergence in the correlated traits of T. pratense cultivars and wild populations that could lead to outbreeding depression if the plants interbreed. PMID:27552193

  2. [Genetic screening and analysis of suppressors of asa1-1 (soa) defective in jasmonate-mediated lateral root formation in Arabidopsis].

    PubMed

    Li, Yan-An; Qi, Lin-Lin; Sun, Jia-Qiang; Liu, Hong-Yu; Li, Chuan-You

    2011-09-01

    It has been shown that jasmonate modulates the lateral root development through crosstalk with auxin in Arabidopsis thaliana. Exogenous application of jasmonate stimulates lateral root formation in wild type but inhibits lateral root formation in asa1-1. Our previous work has demonstrated that the lateral root formation defect of asa1-1 is co-related with jasmonte effect on PIN2 protein levels. To further elucidate the molecular mechanisms underlying jasmonate-mediated reduction of plasma membrane (PM)-resident PIN2 abundance, we have conducted a genetic screen to identify suppressors of asa1-1 (soa), which showed lateral root formation in the presence of jasmonate. Here, we described the basic characterization of soa563 and soa856. We showed that both soa563 and soa856 displayed restored lateral root formation in response to exogenous jasmonate. In addition, jasmonate-induced PIN2:GFP reduction was blocked in these two mutants. Our on-going effort to identify genes defined by these mutants promise to shed new light on the understanding of the molecular mechanisms controlling jasmonate-mediated regulation of PIN2 protein trafficking and turnover.

  3. The reduced insulin-mediated glucose oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin--evidence from cultured myotubes.

    PubMed

    Gaster, Michael; Beck-Nielsen, Henning

    2004-09-06

    Several defects in response to insulin have been described in vivo and in vitro in type 2 diabetes: a decreased glucose transport, defective glucose oxidation and altered glycogen synthesis. At present, it is unknown whether glucose oxidation is primarily affected or secondarily affected by, e.g. increased free fatty acids (FFA). The aim of this study was to evaluate whether myotubes established from type 2 diabetic subjects express a primarily or a FFA-induced reduced insulin-mediated glucose oxidation. We have therefore investigated glucose oxidation under basal, physiological conditions and during acute insulin stimulation with/without FFA. We found that myotubes established from type 2 diabetic subjects express a reduced insulin-stimulated increase in glucose oxidation. Moreover, an acute exposure to FFA reduces insulin-mediated glucose oxidation without alterations in glucose uptake and glycogen synthesis. Thus, we conclude that the diminished increase in insulin-stimulated glucose oxidation seen in type 2 diabetic subjects in vivo may be of genetic origin. Moreover, the glucose-fatty acid cycle seems not to be crucial for the pathophysiology of insulin resistance.

  4. Meta-Analysis of Global Transcriptomics Suggests that Conserved Genetic Pathways are Responsible for Quercetin and Tannic Acid Mediated Longevity in C. elegans

    PubMed Central

    Pietsch, Kerstin; Saul, Nadine; Swain, Suresh C.; Menzel, Ralph; Steinberg, Christian E. W.; Stürzenbaum, Stephen R.

    2012-01-01

    Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of Caenorhabditis elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to three concentrations of Quercetin or Tannic acid, respectively. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published datasets derived from (i) longevity mutants or (ii) infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the prediction of putative mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling, and the p38 MAPK pathway and Tannic acid’s impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, and genetic players of the p38 MAPK pathway therefore seem to be crucial regulators for both polyphenols. Taken together, this study underlines how meta-analyses can provide an insight of molecular events that go beyond the traditional categorization into gene ontology-terms and Kyoto encyclopedia of genes and genomes-pathways. It also supports the call to expand the generation of comparative and integrative databases, an effort that is currently still in its infancy. PMID:22493606

  5. Meta-Analysis of Global Transcriptomics Suggests that Conserved Genetic Pathways are Responsible for Quercetin and Tannic Acid Mediated Longevity in C. elegans.

    PubMed

    Pietsch, Kerstin; Saul, Nadine; Swain, Suresh C; Menzel, Ralph; Steinberg, Christian E W; Stürzenbaum, Stephen R

    2012-01-01

    Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of Caenorhabditis elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to three concentrations of Quercetin or Tannic acid, respectively. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published datasets derived from (i) longevity mutants or (ii) infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the prediction of putative mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling, and the p38 MAPK pathway and Tannic acid's impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, and genetic players of the p38 MAPK pathway therefore seem to be crucial regulators for both polyphenols. Taken together, this study underlines how meta-analyses can provide an insight of molecular events that go beyond the traditional categorization into gene ontology-terms and Kyoto encyclopedia of genes and genomes-pathways. It also supports the call to expand the generation of comparative and integrative databases, an effort that is currently still in its infancy.

  6. In Vitro Acid-Mediated Initial Dental Enamel Loss Is Associated with Genetic Variants Previously Linked to Caries Experience.

    PubMed

    Vieira, Alexandre R; Bayram, Merve; Seymen, Figen; Sencak, Regina C; Lippert, Frank; Modesto, Adriana

    2017-01-01

    We have previously shown that AQP5 and BTF3 genetic variation and expression in whole saliva are associated with caries experience suggesting that these genes may have a functional role in protecting against caries. To further explore these results, we tested ex vivo if variants in these genes are associated with subclinical dental enamel mineral loss. DNA and enamel samples were obtained from 53 individuals. Enamel samples were analyzed for Knoop hardness of sound enamel, integrated mineral loss after subclinical carious lesion creation, and change in integrated mineral loss after remineralization. DNA samples were genotyped for single nucleotide polymorphisms using TaqMan chemistry. Chi-square and Fisher's exact tests were used to compare individuals above and below the mean sound enamel microhardness of the cohort with alpha of 0.05. The A allele of BTF3 rs6862039 appears to be associated with harder enamel at baseline (p = 0.09), enamel more resistant to demineralization (p = 0.01), and enamel that more efficiently regain mineral and remineralize (p = 0.04). Similarly, the G allele of AQP5 marker rs3759129 and A allele of AQP5 marker rs296763 are associated with enamel more resistant to demineralization (p = 0.03 and 0.05, respectively). AQP5 and BTF3 genetic variations influence the initial subclinical stages of caries lesion formation in the subsurface of enamel.

  7. In Vitro Acid-Mediated Initial Dental Enamel Loss Is Associated with Genetic Variants Previously Linked to Caries Experience

    PubMed Central

    Vieira, Alexandre R.; Bayram, Merve; Seymen, Figen; Sencak, Regina C.; Lippert, Frank; Modesto, Adriana

    2017-01-01

    We have previously shown that AQP5 and BTF3 genetic variation and expression in whole saliva are associated with caries experience suggesting that these genes may have a functional role in protecting against caries. To further explore these results, we tested ex vivo if variants in these genes are associated with subclinical dental enamel mineral loss. DNA and enamel samples were obtained from 53 individuals. Enamel samples were analyzed for Knoop hardness of sound enamel, integrated mineral loss after subclinical carious lesion creation, and change in integrated mineral loss after remineralization. DNA samples were genotyped for single nucleotide polymorphisms using TaqMan chemistry. Chi-square and Fisher's exact tests were used to compare individuals above and below the mean sound enamel microhardness of the cohort with alpha of 0.05. The A allele of BTF3 rs6862039 appears to be associated with harder enamel at baseline (p = 0.09), enamel more resistant to demineralization (p = 0.01), and enamel that more efficiently regain mineral and remineralize (p = 0.04). Similarly, the G allele of AQP5 marker rs3759129 and A allele of AQP5 marker rs296763 are associated with enamel more resistant to demineralization (p = 0.03 and 0.05, respectively). AQP5 and BTF3 genetic variations influence the initial subclinical stages of caries lesion formation in the subsurface of enamel. PMID:28275354

  8. Congenic mice provide in vivo evidence for a genetic locus that modulates intrinsic transforming growth factor β1-mediated signaling and bone acquisition.

    PubMed

    Mukherjee, Aditi; Larson, Emily A; Carlos, Amy S; Belknap, John K; Rotwein, Peter; Klein, Robert F

    2012-06-01

    Osteoporosis, the most common skeletal disorder, is characterized by low bone mineral density (BMD) and an increased risk of fragility fractures. BMD is the best clinical predictor of future osteoporotic fracture risk, but is a complex trait controlled by multiple environmental and genetic determinants with individually modest effects. Quantitative trait locus (QTL) mapping is a powerful method for identifying chromosomal regions encompassing genes involved in shaping complex phenotypes, such as BMD. Here we have applied QTL analysis to male and female genetically-heterogeneous F(2) mice derived from a cross between C57BL/6 and DBA/2 strains, and have identified 11 loci contributing to femoral BMD. Further analysis of a QTL on mouse chromosome 7 following the generation of reciprocal congenic strains has allowed us to determine that the high BMD trait, which tracks with the DBA/2 chromosome and exerts equivalent effects on male and female mice, is manifested by enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and by increased growth of metatarsal bones in short-term primary culture. An insertion/deletion DNA polymorphism in Ltbp4 exon 12 that causes the in-frame removal of 12 codons in the DBA/2-derived gene maps within 0.6 Mb of the marker most tightly linked to the QTL. LTBP4, one of four paralogous mouse proteins that modify the bioavailability of the transforming growth factor β (TGF-β) family of growth factors, is expressed in differentiating MSC-derived osteoblasts and in long bones, and reduced responsiveness to TGF-β1 is observed in MSCs of mice homozygous for the DBA/2 chromosome 7. Taken together, our results identify a potential genetic and biochemical relationship between decreased TGF-β1-mediated signaling and enhanced femoral BMD that may be regulated by a variant LTBP4 molecule.

  9. Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

    PubMed Central

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-01-01

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field. PMID:25167136

  10. Development and application of loop-mediated isothermal amplification assays for rapid visual detection of cry2Ab and cry3A genes in genetically-modified crops.

    PubMed

    Li, Feiwu; Yan, Wei; Long, Likun; Qi, Xing; Li, Congcong; Zhang, Shihong

    2014-08-27

    The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.

  11. Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii.

    PubMed

    Giuliano Garisto Donzelli, Bruno; Krasnoff, Stuart B; Moon, Yong-Sun; Sun-Moon, Yong; Churchill, Alice C L; Gibson, Donna M

    2012-04-01

    Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-L: -valine and N-methyl-L: -alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.

  12. Marrow-dependent cells depleted by 89Sr mediate genetic resistance to herpes simplex virus type 1 infection in mice.

    PubMed

    Lopez, C; Ryshke, R; Bennett, M

    1980-06-01

    Adult mice resistant to infection with 10(6) plaque-forming units of a virulent strain of herpes simplex virus type 1 were treated with 89Sr to abrogate marrow-dependent cell functions. Treated mice were found to be much more susceptible to the herpes simplex virus type 1 infection than untreated mice. The virus persisted in the visceral tissues of 89Sr-treated mice for 3 or more days postinfection but not in those of untreated mice. The virus also spread to the spinal cords of treated but not untreated mice. A marrow-dependent cell appeared to mediate resistance to herpes simplex virus type 1 by controlling the infection early after inoculation and not allowing the infection spread to the central nervous system.

  13. Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids

    PubMed Central

    Frost, John R.; Jacob, Nicholas T.; Papa, Louis J.; Owens, Andrew E.

    2015-01-01

    A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles. PMID:25933125

  14. A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

    PubMed

    Metcalf, W W; Zhang, J K; Apolinario, E; Sowers, K R; Wolfe, R S

    1997-03-18

    New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.

  15. Use of recombination-mediated genetic engineering for construction of rescue human cytomegalovirus bacterial artificial chromosome clones.

    PubMed

    Dulal, Kalpana; Silver, Benjamin; Zhu, Hua

    2012-01-01

    Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  16. Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram †

    PubMed Central

    Bush, Arla L.; Pueppke, Steven G.

    1991-01-01

    We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6. Images PMID:16348549

  17. Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium.

    PubMed

    Gallie, D R; Novak, S; Kado, C I

    1985-09-01

    Presented are a set of cosmids based on the unit copy Agrobacterium plasmid, pTAR, and the high-copy-number mutant plasmid, pUCD500, of pTiC58. The addition of a par function derived from pTAR to the vectors allowed them to be stably maintained throughout the cell population in the absence of selective pressure. These vectors, designed for Agrobacterium and Rhizobium, also work in Escherichia coli. The vectors can be cotransferred to Rhizobiaceae from E. coli with the helper plasmid, pRK2013. The pTiC58 origin containing vectors, pUCD1000 and pUCD1001 were found to be incompatible with a 250-kb plasmid harbored by R. meliloti RM102Z1. RM102Z1(pUCD1000) was still capable of nodulating roots in alfalfa.

  18. Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules.

    PubMed

    Holme, Inger Baeksted; Brinch-Pedersen, Henrik; Lange, Mette; Holm, Preben Bach

    2006-12-01

    We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.

  19. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

    PubMed Central

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J.; Sarkar, Mayukh K.; Li, Feng

    2015-01-01

    ABSTRACT Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCE For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are

  20. Isolation and Characterization of Agrobacterium Strains from Soil: A Laboratory Capstone Experience†

    PubMed Central

    Finer, Kim R.; Fox, Lee; Finer, John J.

    2016-01-01

    In this investigation, the students’ goal was to isolate and characterize Agrobacterium strains from soil. Following selection and enrichment on 1A-t medium, putative Agrobacterium isolates were characterized by Gram stain reaction and biochemical tests. Isolates were further evaluated using polymerase chain reaction (PCR) with different primer sets designed to amplify specific regions of bacterial deoxyribonucleic acid (DNA). Primer sets included AGRH to identify isolates that were members of the Rhizobiaceae, BIOVAR1 primers to identify members of Agrobacterium biovar group I, and a third set, VIRG, to determine presence of virG (only present in pathogenic Agrobacterium strains). During the investigation, students applied previously learned techniques including serial dilution, use of selective/differential media, staining protocols, biochemical analysis, molecular analysis via PCR, and electrophoresis. Students also gained practical experience using photo documentation to record data for an eventual mock journal publication of the capstone laboratory experience. Pre- and post-evaluation of class content knowledge related to the techniques, protocols, and learning objectives of these laboratories revealed significant learning gains in the content areas of Agrobacterium–plant interactions (p ≤ 0.001) and molecular biology (p ≤ 0.01). The capstone journal assignment served as the assessment tool to evaluate mastery and application of laboratory technique, the ability to accurately collect and evaluate data, and critical thinking skills associated with experimental troubleshooting and extrapolation. Analysis of journal reports following the capstone experience showed significant improvement in assignment scores (p ≤ 0.0001) and attainment of capstone experience learning outcomes. PMID:28101272

  1. Aspergillus fumigatus spore proteomics and genetics reveal that VeA represses DefA-mediated DNA damage response.

    PubMed

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young; Heo, In-Beom; Kim, Young Hwan; Yu, Jae-Hyuk

    2016-10-04

    Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues.

  2. Therapeutic hotline. Genetic variations in the androgen receptor gene and finasteride response in women with androgenetic alopecia mediated by epigenetics.

    PubMed

    Keene, Sharon; Goren, Andy

    2011-01-01

    When studies of postmenopausal women with hair loss failed to reveal a response to the 5 alpha reductase inhibitor, finasteride, researchers began to question the existence of androgenetic alopecia in women and renamed the clinical entity female pattern hair loss. However, recently published reports of finasteride response in some women with hair loss suggest that an androgenic mechanism is mediating response in this group. Variant repeat nucleotide sequences in exon 1 of the androgen receptor (AR) gene have been shown to determine androgen sensitivity in a variety of androgenic conditions in men and women. In an effort to identify whether this AR variant may help determine which women are likely to respond to finasteride therapy, a pilot study was undertaken. In our 6-month pilot of 13 patients, women with greater androgen sensitivity (<24 cytosine, adenine, and guanine (CAG) repeats) were likely to have a significant response to finasteride 1 mg/day compared with patients treated with placebo, and compared with patients with normal androgen sensitivity (≥24 CAG repeats) based on epigenetic weighted evaluation of the CAG alleles. Results of the present pilot study support the hypothesis that AR-CAG repeats, in conjunction with epigenetic factors, can help determine which women with hair loss will respond to finasteride therapy.

  3. Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension

    PubMed Central

    Bencze, Michal; Vaněčková, Ivana; Kuneš, Jaroslav; Zicha, Josef

    2017-01-01

    Calcium sensitization mediated by RhoA/Rho kinase pathway can be evaluated either in the absence (basal calcium sensitization) or in the presence of endogenous vasoconstrictor systems (activated calcium sensitization). Our aim was to compare basal and activated calcium sensitization in three forms of experimental hypertension with increased sympathetic tone and enhanced calcium entry—spontaneously hypertensive rats (SHR), heterozygous Ren-2 transgenic rats (TGR), and salt hypertensive Dahl rats. Activated calcium sensitization was determined as blood pressure reduction induced by acute administration of Rho kinase inhibitor fasudil in conscious rats with intact sympathetic nervous system (SNS) and renin-angiotensin system (RAS). Basal calcium sensitization was studied as fasudil-dependent difference in blood pressure response to calcium channel opener BAY K8644 in rats subjected to RAS and SNS blockade. Calcium sensitization was also estimated from reduced development of isolated artery contraction by Rho kinase inhibitor Y-27632. Activated calcium sensitization was enhanced in all three hypertensive models (due to the hyperactivity of vasoconstrictor systems). In contrast, basal calcium sensitization was reduced in SHR and TGR relative to their controls, whereas it was augmented in salt-sensitive Dahl rats relative to their salt-resistant controls. Similar differences in calcium sensitization were seen in femoral arteries of SHR and Dahl rats. PMID:28197417

  4. Basal and Activated Calcium Sensitization Mediated by RhoA/Rho Kinase Pathway in Rats with Genetic and Salt Hypertension.

    PubMed

    Behuliak, Michal; Bencze, Michal; Vaněčková, Ivana; Kuneš, Jaroslav; Zicha, Josef

    2017-01-01

    Calcium sensitization mediated by RhoA/Rho kinase pathway can be evaluated either in the absence (basal calcium sensitization) or in the presence of endogenous vasoconstrictor systems (activated calcium sensitization). Our aim was to compare basal and activated calcium sensitization in three forms of experimental hypertension with increased sympathetic tone and enhanced calcium entry-spontaneously hypertensive rats (SHR), heterozygous Ren-2 transgenic rats (TGR), and salt hypertensive Dahl rats. Activated calcium sensitization was determined as blood pressure reduction induced by acute administration of Rho kinase inhibitor fasudil in conscious rats with intact sympathetic nervous system (SNS) and renin-angiotensin system (RAS). Basal calcium sensitization was studied as fasudil-dependent difference in blood pressure response to calcium channel opener BAY K8644 in rats subjected to RAS and SNS blockade. Calcium sensitization was also estimated from reduced development of isolated artery contraction by Rho kinase inhibitor Y-27632. Activated calcium sensitization was enhanced in all three hypertensive models (due to the hyperactivity of vasoconstrictor systems). In contrast, basal calcium sensitization was reduced in SHR and TGR relative to their controls, whereas it was augmented in salt-sensitive Dahl rats relative to their salt-resistant controls. Similar differences in calcium sensitization were seen in femoral arteries of SHR and Dahl rats.

  5. Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease

    PubMed Central

    Ventham, N. T.; Kennedy, N. A.; Adams, A. T.; Kalla, R.; Heath, S.; O'Leary, K. R.; Drummond, H.; Lauc, Gordan; Campbell, Harry; McGovern, Dermot P. B.; Annese, Vito; Zoldoš, Vlatka; Permberton, Iain K.; Wuhrer, Manfred; Kolarich, Daniel; Fernandes, Daryl L.; Theorodorou, Evropi; Merrick, Victoria; Spencer, Daniel I.; Gardner, Richard A.; Doran, Ray; Shubhakar, Archana; Boyapati, Ray; Rudan, Igor; Lionetti, Paolo; Akmačić, Irena Trbojević; Krištić, Jasminka; Vučković, Frano; Štambuk, Jerko; Novokmet, Mislav; Pučić-Baković, Maja; Gornik, Olga; Andriulli, Angelo; Cantoro, Laura; Sturniolo, Giancarlo; Fiorino, Gionata; Manetti, Natalia; Latiano, Anna; Kohn, Anna; D'Incà, Renata; Danese, Silvio; Arnott, Ian D.; Noble, Colin L.; Lees, Charlie W.; Shand, Alan G.; Ho, Gwo-Tzer; Dunlop, Malcolm G.; Murphy, Lee; Gibson, Jude; Evenden, Louise; Wrobel, Nicola; Gilchrist, Tamara; Fawkes, Angie; Kammeijer, Guinevere S. M.; Clerc, Florent; de Haan, Noortje; Vojta, Aleksandar; Samaržija, Ivana; Markulin, Dora; Klasić, Marija; Dobrinić, Paula; Aulchenko, Yurii; van den Heuve, Tim; Jonkers, Daisy; Pierik, Marieke; Vatn, Simen; Ricanek, Petr; Jahnsen, Jørgen; You, Panpan; Sølvernes, Janne; Frengen, Anna B.; Tannæs, Tone M.; Moen, Aina E. F.; Dahl, Fredrik A.; Lindstrøm, Jonas Christoffer; Ekeland, Gunn S.; Detlie, Trond Espen; Keita, Åsa V.; Söderholm, Johan D.; Hjortswang, Henrik; Halfvarson, Jonas; Bergemalm, Daniel; Gomollón, Fernando; D'Amato, Mauro; Törkvist, Leif; Hjelm, Fredrik; Gullberg, Mats; Nordberg, Niklas; Ocklind, Anette; Pettersson, Erik; Ekman, Daniel; Sundell, Mikael; Modig, Eddie; Veillard, Anne- Clémence; Schoemans, Renaud; Poncelet, Dominique; Sabatel, Céline; Gut, Marta; Bayes, Monica; Casén, Christina; Lindahl, Torbjørn; Ciemniejewska, Ewa; Vatn, Morten H.; Wilson, D. C.; Gut, I. G.; Nimmo, E. R.; Satsangi, J.

    2016-01-01

    Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. PMID:27886173

  6. Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease.

    PubMed

    Ventham, N T; Kennedy, N A; Adams, A T; Kalla, R; Heath, S; O'Leary, K R; Drummond, H; Wilson, D C; Gut, I G; Nimmo, E R; Satsangi, J

    2016-11-25

    Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8(+) T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression.

  7. Genetic evidence reveals density-dependent mediated success of alternative mating behaviours in the European bitterling (Rhodeus sericeus).

    PubMed

    Reichard, M; Smith, C; Jordan, W C

    2004-06-01

    The reproductive success of alternative mating behaviours may vary within and among populations in relation to environmental factors and demographic parameters. We used behavioural and genetic data to investigate how male density affects reproductive success of territoriality and sneaking in the European bitterling (Rhodeus sericeus, Cyprinidae), a freshwater fish that spawns on the gills of living freshwater mussels. Keeping the number of spawning sites constant, we manipulated male densities in laboratory and mesocosm experiments. We showed that sneaked fertilizations were common in R. sericeus, and that they increased significantly with male density. Territorial mating was almost 17 times more successful than sneaking at the lowest male density treatment, and still 2-3 times more successful at intermediate densities. However, both behaviours conferred the same fitness pay-off at the highest male density. While the success of territorial males declined with male density, the success of individual sneaking males remained constant across densities. Notably, the capacity of territorial males to outcompete sneakers by preoviposition sperm loading was the best predictor of male reproductive success, rather than aggression, body size or postoviposition ejaculation.

  8. Genetic characterization of plasmid-mediated quinolone resistance gene qnrS2 in Pseudoalteromonas and Shewanella isolates from seawater.

    PubMed

    Zhao, Jing-Yi; Zhao, Shu-Mei; Mu, Xiao-Dong; Xiao, Zijun

    2016-12-23

    Three qnrS2-containing isolates of Pseudoalteromonas and Shewanella were collected from the seawater samples of Qingdao in China during 2014. They displayed resistance to ampicillin, ciprofloxacin, kanamycin, nalidixic acid and sulfamethoxazole. The qnrS2 genes were identified in the chromosomes of Pseudoalteromonas strain E8 and S16, and in a 140-kb plasmid in Shewanella strain S14, respectively. In addition, two copies of qnrS2 were identified in the strain E8. Sequence analyses revealed that there was an identical DNA segment located in the downstream of qnrS2 in strain S14 and E8, coding for a TetR transcriptional regulator, two putative integrases and a hypothetical protein. However, different genetic structures were identified in the upstream sequences: the terB gene associated with tellurite resistance in the strain S14, and a putative integron with dfrA6 and aadA13 gene cassettes or the Tn7-related gene complex tnsABC in the strain E8. In Pseudoalteromonas strain S16, qnrS2 was bracketed by the endonuclease I and III genes, and the electron transport complex rsxCDGE was located in the upstream sequences. This is the first report of two copies of the qnrS2 gene existing in one bacterial chromosome, and also the first identification of qnrS2 in Shewanella.

  9. Progress in genetic engineering of peanut (Arachis hypogaea L.)--a review.

    PubMed

    Krishna, Gaurav; Singh, Birendra K; Kim, Eun-Ki; Morya, Vivek K; Ramteke, Pramod W

    2015-02-01

    Peanut (Arachis hypogaea L.) is a major species of the family, Leguminosae, and economically important not only for vegetable oil but as a source of proteins, minerals and vitamins. It is widely grown in the semi-arid tropics and plays a role in the world agricultural economy. Peanut production and productivity is constrained by several biotic (insect pests and diseases) and abiotic (drought, salinity, water logging and temperature aberrations) stresses, as a result of which crop experiences serious economic losses. Genetic engineering techniques such as Agrobacterium tumefaciens and DNA-bombardment-mediated transformation are used as powerful tools to complement conventional breeding and expedite peanut improvement by the introduction of agronomically useful traits in high-yield background. Resistance to several fungal, virus and insect pest have been achieved through variety of approaches ranging from gene coding for cell wall component, pathogenesis-related proteins, oxalate oxidase, bacterial chloroperoxidase, coat proteins, RNA interference, crystal proteins etc. To develop transgenic plants withstanding major abiotic stresses, genes coding transcription factors for drought and salinity, cytokinin biosynthesis, nucleic acid processing, ion antiporter and human antiapoptotic have been used. Moreover, peanut has also been used in vaccine production for the control of several animal diseases. In addition to above, this study also presents a comprehensive account on the influence of some important factors on peanut genetic engineering. Future research thrusts not only suggest the use of different approaches for higher expression of transgene(s) but also provide a way forward for the improvement of crops.

  10. Increase in SGLT1-mediated transport explains renal glucose reabsorption during genetic and pharmacological SGLT2 inhibition in euglycemia.

    PubMed

    Rieg, Timo; Masuda, Takahiro; Gerasimova, Maria; Mayoux, Eric; Platt, Kenneth; Powell, David R; Thomson, Scott C; Koepsell, Hermann; Vallon, Volker

    2014-01-01

    In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90 and ∼3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40-50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1-/-) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5- to 2-fold higher urine glucose/creatinine ratios in Sglt1-/- vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1-/- vs. WT after 24 h (-33 vs. -11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 ± 2% in WT and 17 ± 2% in Sglt1-/-. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ∼10-fold and reduced FGR to 44 ± 3% in WT and to -1 ± 3% in Sglt1-/-. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50-60% of filtered glucose is excreted.

  11. Intratumoral mediated immunosuppression is prognostic in genetically engineered murine models of glioma and correlates to immune therapeutic responses

    PubMed Central

    Kong, Ling-Yuan; Wu, Adam S.; Doucette, Tiffany; Wei, Jun; Priebe, Waldemar; Fuller, Gregory N.; Qiao, Wei; Sawaya, Raymond; Rao, Ganesh; Heimberger, Amy B.

    2010-01-01

    Purpose Pre-clinical murine model systems used for the assessment of therapeutics have not been predictive of human clinical responses, primarily because their clonotypic nature does not recapitulate the heterogeneous biology and immunosuppressive mechanisms of humans. Relevant model systems with mice that are immunologically competent are needed to evaluate the efficacy of therapeutic agents, especially immunotherapeutics. Experimental Design Using the RCAS/Ntv-a system, mice were engineered to co-express platelet-derived growth factor receptor (PDGF)-B + B-cell lymphoma (Bcl)-2 under the control of the glioneuronal-specific Nestin promoter. The degree and type of tumor-mediated immunosuppression was determined in these endogenously arising gliomas based upon the presence of macrophages and regulatory T cells (Tregs). The immunotherapeutic agent, WP1066, was tested in vivo to assess therapeutic efficacy and immune modulation. Results N-tva mice were injected with RCAS vectors to express PDGF-B + Bcl-2, resulting in both low- and high-grade gliomas. Consistent with observations in human high-grade gliomas, mice with high-grade gliomas also developed a marked intratumoral influx of macrophages that was influenced by tumor signal transducer and activator of transduction (STAT) 3 expression. The presence of intratumoral F4/80 macrophages was a negative prognosticator for long-term survival. In mice expressing both PDGF-B + Bcl-2 that were treated with WP1066, there was 55.5% increase in median survival time (P< 0.01), with an associated inhibition of intratumoral STAT3 and macrophages. Conclusions Although randomization is necessary for including mice in a therapeutic trial, these murine model systems are more suitable for testing therapeutics, and especially immune therapeutics, in the context of translational studies. PMID:20921210

  12. Genetic vaccines to potentiate the effective CD103+ dendritic cell-mediated cross-priming of antitumor immunity.

    PubMed

    Zhang, Yi; Chen, Guo; Liu, Zuqiang; Tian, Shenghe; Zhang, Jiying; Carey, Cara D; Murphy, Kenneth M; Storkus, Walter J; Falo, Louis D; You, Zhaoyang

    2015-06-15

    The development of effective cancer vaccines remains an urgent, but as yet unmet, clinical need. This deficiency is in part due to an incomplete understanding of how to best invoke dendritic cells (DC) that are crucial for the induction of tumor-specific CD8(+) T cells capable of mediating durable protective immunity. In this regard, elevated expression of the transcription factor X box-binding protein 1 (XBP1) in DC appears to play a decisive role in promoting the ability of DC to cross-present Ags to CD8(+) T cells in the therapeutic setting. Delivery of DNA vaccines encoding XBP1 and tumor Ag to skin DC resulted in increased IFN-α production by plasmacytoid DC (pDC) from skin/tumor draining lymph nodes and the cross-priming of Ag-specific CD8(+) T cell responses associated with therapeutic benefit. Antitumor protection was dependent on cross-presenting Batf3(+) DC, pDC, and CD8(+) T cells. CD103(+) DC from the skin/tumor draining lymph nodes of the immunized mice appeared responsible for activation of Ag-specific naive CD8(+) T cells, but were dependent on pDC for optimal effectiveness. Similarly, human XBP1 improved the capacity of human blood- and skin-derived DC to activate human T cells. These data support an important intrinsic role for XBP1 in DC for effective cross-priming and orchestration of Batf3(+) DC-pDC interactions, thereby enabling effective vaccine induction of protective antitumor immunity.

  13. Zebrafish blowout provides genetic evidence for Patched1-mediated negative regulation of Hedgehog signaling within the proximal optic vesicle of the vertebrate eye.

    PubMed

    Lee, Jiwoon; Willer, Jason R; Willer, Gregory B; Smith, Kierann; Gregg, Ronald G; Gross, Jeffrey M

    2008-07-01

    In this study, we have characterized the ocular defects in the recessive zebrafish mutant blowout that presents with a variably penetrant coloboma phenotype. blowout mutants develop unilateral or bilateral colobomas and as a result, the retina and retinal pigmented epithelium are not contained within the optic cup. Colobomas result from defects in optic stalk morphogenesis whereby the optic stalk extends into the retina and impedes the lateral edges of the choroid fissure from meeting and fusing. The expression domain of the proximal optic vesicle marker pax2a is expanded in blowout at the expense of the distal optic vesicle marker pax6, suggesting that the initial patterning of the optic vesicle into proximal and distal territories is disrupted in blowout. Later aspects of distal optic cup formation (i.e. retina development) are normal in blowout mutants, however. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway, as the underlying cause of the blowout phenotype. Expanded domains of expression of the Hedgehog target genes patched1 and patched2 were observed in blowout, consistent with a loss of Patched1 function and upregulation of Hedgehog pathway activity. Moreover, colobomas in blowout could be suppressed by pharmacologically inhibiting the Hedgehog pathway with cyclopamine, and maximal rescue occurred when embryos were exposed to cyclopamine between 5.5 and 13 hours post-fertilization. These observations highlight the critical role that Hedgehog pathway activity plays in mediating patterning of the proximal/distal axis of the optic vesicle during the early phases of eye development and they provide genetic confirmation for the integral role that patched1-mediated negative regulation of Hedgehog signaling plays during vertebrate eye development.

  14. Genetically Altered Mutant Mouse Models of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Exhibit the Cardiac Expression of Proinflammatory Mediators in a Gene-Dose-Dependent Manner

    PubMed Central

    Vellaichamy, Elangovan; Das, Subhankar; Subramanian, Umadevi; Maeda, Nobuyo

    2014-01-01

    The objective of this study was to examine whether genetically determined differences in the guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1) affect cardiac expression of proinflammatory cytokines, hypertrophic markers, nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) in am Npr1 gene-dose–dependent manner. In the present studies, adult male Npr1 gene-disrupted (Npr1−/−), wild-type (Npr1+/+), and gene-duplicated (Npr1++/++) mice were used. The Npr1−/− mice showed 41 mm Hg higher systolic blood pressure and 60% greater heart weight to body weight (HW/BW) ratio; however, Npr1++/++ mice exhibited 15 mm Hg lower systolic blood pressure and 12% reduced HW/BW ratio compared with Npr1+/+ mice. Significant upregulation of gene expression of proinflammatory cytokines and hypertrophic markers along with enhanced NF-κB/AP-1 binding activities were observed in the Npr1−/− mouse hearts. Conversely, hypertrophic markers and proinflammatory cytokines gene expression as well as NF-κB/AP-1 binding activities were markedly decreased in Npr1++/++ mouse hearts compared with wild-type mice. The ventricular guanylyl cyclase activity and cGMP levels were reduced by 96% and 87%, respectively, in Npr1−/− mice; however, these parameters were amplified by 2.8-fold and 3.8-fold, respectively, in Npr1++/++ mice. Echocardiographic analysis revealed significantly increased fractional shortening in Npr1++/++ mice (P < .05) but greatly decreased in Npr1−/− mice (P < .01) hearts compared with Npr1+/+ mice. The present findings suggest that Npr1 represses the expression of cardiac proinflammatory mediators, hypertrophic markers, and NF-κB/AP-1–mediated mechanisms, which seem to be associated in an Npr1 gene-dose–dependent manner. PMID:24424043

  15. Early post-myocardial infarction survival in MRL mice is mediated by attenuated apoptosis and inflammation but depends on genetic background

    PubMed Central

    Hunt, Darlene L.; Campbell, Patrick H.; Zambon, Alexander C.; Vranizan, Karen; Evans, Sylvia M.; Kuo, Hai-Chien; Yamaguchi, Ken D.; Omens, Jeffrey H.; McCulloch, Andrew D.

    2011-01-01

    The Murphy Roths Large (MRL) mouse, a strain capable of regenerating right ventricular myocardium, has a high post-myocardial infarction (MI) survival rate compared with C57BL/6J (C57) mice. The biological processes responsible for this survival advantage are unknown. To assess the effect of genetic background, the LG/J strain, which harbors 75% of the MRL composite genome, was included in the study. The MRL survival advantage versus C57 mice (92% vs. 68%, P < 0.05) occurred primarily in the first 5 days; LG/J survival was intermediate (P = NS). Microarray data analysis revealed an attenuation of apoptotic (P < 0.05) and stress response transcripts in MRL hearts compared with C57 hearts after MI. Supporting the microarray results, there were fewer TUNEL-positive cells 1 day post-MI in MRL infarcts compared with C57 infarcts (P = 0.001) and fewer CD45-positive cells in the MRL infarct border zone 2 days post-MI (P < 0.01). LG/J results were intermediate (P = NS). MRL hearts had smaller infarct scars and attenuated ventricular dilation 30 days post-MI compared with C57 hearts (P < 0.05). We conclude that the early post-MI survival advantage of MRL mice over the C57 strain is mediated at least in part by reductions in apoptosis and infla