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Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Masafumi Mikami, Masaki Endo, Sakiko Hirose, Seiichi Toki, Akito Kaga, Ken Naito, Masao Ishimoto, Jun Abe, Tetsuya Yamada
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD)...
January 15, 2018: Plant Cell Reports
Magdalena Klimek-Chodacka, Tomasz Oleszkiewicz, Levi G Lowder, Yiping Qi, Rafal Baranski
The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome...
January 13, 2018: Plant Cell Reports
Jie Zhao, Shenquan Zeng, Ying Xia, Liming Xia
The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium-mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei. Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei. Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8...
January 10, 2018: Journal of Bioscience and Bioengineering
Galina Khafizova, Pavel Dobrynin, Dmitrii Polev, Tatiana Matveeva
OBJECTIVE: Nicotiana glauca (tree tobacco) is a naturally transgenic plant, containing sequences acquired from Agrobacterium rhizogenes by horizontal gene transfer. Besides, N. glauca contains a wide profile of alkaloids of medical interest. DATA DESCRIPTION: We report a high-depth sequencing and de novo assembly of N. glauca full genome and analysis of genome elements with bacterial origin. The draft genome assembly is 3.2 Gb, with N50 size of 31.1 kbp. Comparative analysis confirmed the presence of single, previously described gT insertion...
January 12, 2018: BMC Research Notes
Ping Che, Ajith Anand, Emily Wu, Jeffry D Sander, Marissa K Simon, Weiwei Zhu, Amy L Sigmund, Gina Zastrow-Hayes, Michael Miller, Donglong Liu, Shai J Lawit, Zuo-Yu Zhao, Marc C Albertsen, Todd J Jones
Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here we report a ternary vector (also known as co-habitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6-29% in Tx430 using different selectable markers and single copy, backbone free "quality events" ranging from 45-66% of the total events produced...
January 11, 2018: Plant Biotechnology Journal
Shengyan Li, Zeyu Wang, Yiyao Zhou, Changhui Li, Guiping Wang, Hai Wang, Jie Zhang, Gemei Liang, Zhihong Lang
To improve the novel Bacillus thuringiensis insecticidal gene cry2Ah1 toxicity, two mutants cry2Ah1-vp (V354VP) and cry2Ah1-sp (V354SP) were performed. SWISS-MODEL analysis showed two mutants had a longer loop located between β-4 and β-5 of domain II, resulting in higher binding affinity with brush border membrane vesicles (BBMV) of Helicoverpa armigera comparing with Cry2Ah1. The cry2Ah1, cry2Ah1-vp, and cry2Ah1-sp were optimized codon usage according to plant codon bias, and named mcry2Ah1, mcry2Ah1-vp, and mcry2Ah1-sp...
January 11, 2018: Scientific Reports
Shinji Yamamoto, Ayako Sakai, Vita Agustina, Kazuki Moriguchi, Katsunori Suzuki
Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi...
January 9, 2018: Applied Microbiology and Biotechnology
Makoto Hashimoto, Hikaru Kato, Ayako Katsuki, Sachiko Tsukamoto, Isao Fujii
Himeic acid A, produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin-activating enzyme E1 in the ubiquitin-proteasome system. To elucidate the mechanism of himeic acid biosynthesis, we first carried out feeding experiments with labeled precursors. The long fatty acyl side chain attached to the pyrone ring was confirmed to be of polyketide origin, while the amide substituent was found to be derived from leucine. These results suggest that a polyketide synthase-non-ribosomal peptide synthase (PKS-NRPS) is involved in himeic acid biosynthesis...
January 5, 2018: Chembiochem: a European Journal of Chemical Biology
Michelina Ruocco, Riccardo Baroncelli, Santa Olga Cacciola, Catello Pane, Maurilia Maria Monti, Giuseppe Firrao, Mariarosaria Vergara, Gaetano Magnano di San Lio, Giovanni Vannacci, Felice Scala
BACKGROUND: The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D...
January 6, 2018: BMC Genomics
Suzanne M Huddy, Inga I Hitzeroth, Ann E Meyers, Brandon Weber, Edward P Rybicki
Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N...
January 1, 2018: International Journal of Molecular Sciences
Zhenwei Zhang, Liming Liu, Huijie Wu, Lifeng Liu, Baoshan Kang, Bin Peng, Qinsheng Gu
Cucumber green mottle mosaic virus (CGMMV) is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein), followed by the construction of the V94A and T104A mutants...
December 25, 2017: Viruses
Weijia Li, Junxiang Zhang, Hongying Sun, Shouming Wang, Keqin Chen, Yuexue Liu, He Li, Yue Ma, Zhihong Zhang
FveRGA1 was highly expressed in tender tissues such as young leaves and stem apices and was localized in the nucleus. RNAi silencing of FveRGA1 in non-runnering woodland strawberry produced many runners. FveRGA1 is thus a key gene controlling strawberry runner formation. The propagation of strawberry is mainly based on runners, while the genes controlling runner production have not been well characterized. Exogenous applications of optimum concentration gibberellins (GAs) promote runner formation in strawberry cultivation and GA can accelerate the degradation of DELLA proteins...
December 29, 2017: Planta
Khaoula Habbadi, Thibault Meyer, Ludovic Vial, Vincent Gaillard, Rachid Benkirane, Abdellatif Benbouazza, Isabelle Kerzaon, El Hassan Achbani, Céline Lavire
Allorhizobium (Agrobacterium) vitis is a host-specific pathogenic bacterium that causes grapevine crown gall disease, affecting vine growth and production worldwide. The antibacterial activities of different aromatic plant essential oils were tested in vitro and in planta against A. vitis. Among the essential oils tested, those of Origanum compactum and Thymus vulgaris showed the most significant in vitro antibacterial activities, with a MIC of 0.156 and 0.312 mg/mL, respectively. A synergistic effect of these two essential oils (1:1) was observed and confirmed by the checkerboard test...
December 29, 2017: Environmental Science and Pollution Research International
Maheswor Gautam, Mohamed Elhiti, Inge S Fomsgaard
Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound...
December 26, 2017: Chemosphere
Matthew Howell, Jeremy J Daniel, Pamela J B Brown
Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning...
November 24, 2017: Journal of Visualized Experiments: JoVE
Kaiwen Sun, Yuyu Zheng, Ziqiang Zhu
Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions...
November 20, 2017: Journal of Visualized Experiments: JoVE
Shasmita, Manoj K Rai, Soumendra K Naik
Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as "Indian Ginseng", is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves...
December 26, 2017: Critical Reviews in Biotechnology
Qian Liao, Xuan Du, Wei Jiang, Yapei Tong, Zhipeng Zhao, Ruiqin Fang, Juan Feng, Lixia Tang
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential to produce valuable optically pure epoxides and β-substituted alcohols. However, this enzyme has been reported to be very sensitive and less stable under oxidative conditions. Enzyme immobilization represents a powerful means to overcome this limitation and provides the enzyme characteristics of a biocatalyst. In this study, the crude extract of HheC was directly subjected to enzyme immobilization using a carrier-free cross-linked enzyme aggregates (CLEAs) method...
December 19, 2017: Journal of Biotechnology
Tomoyuki Nabeshima, Motoaki Doi, Munetaka Hosokawa
Chrysanthemum stunt viroid (CSVd) was inoculated into two chrysanthemum (Chrysanthemum morifolium) cultivars, the CSVd-susceptible cultivar Piato and the CSVd-resistant cultivar Mari Kazaguruma. For CSVd inoculation, grafting and Agrobacterium-mediated inoculation were used. In grafting experiments, CSVd was detectable in Mari Kazaguruma after grafting onto infected Piato, but after removal of infected rootstocks, CSVd could not be detected in the uppermost leaves. In agroinfection experiments, CSVd systemic infection was observed in Piato but not in Mari Kazaguruma...
2017: Frontiers in Plant Science
Wai Li Lim, Helen M Collins, Rohan R Singh, Natalie Aj Kibble, Kuok Yap, Jillian Taylor, Geoffrey B Fincher, Rachel A Burton
Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however recalcitrance of hull-less cultivars to transformation has limited use of these varieties. To overcome this, we have sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fibre component, (1,3;1,4)-β-glucan (mixed-linkage glucan), primarily found in starchy endosperm cell walls...
December 16, 2017: Journal of Integrative Plant Biology
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