Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.
Agrobacterium ; Agrobacterium tumefaciens ; Fungi ; Insects ; Metarhizium* ; Methods ; Streptothricins ; Virulence

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.
Agrobacterium ; Agrobacterium tumefaciens ; Biology ; Computational Biology ; Cordyceps* ; Electron Transport Complex IV ; Fruit* ; Fungi* ; Gene Ontology ; Telomerase

Rhizobium species, aerobic Gram-negative rods found in soils worldwide, are well-known tumor-inducing pathogens in plants. Since 1980, when the first case of prosthetic valve endocarditis caused by Rhizobium radiobacter was reported, R. radiobacter has been recognized as an opportunistic human pathogen. In Korea, three cases of infection by this organism have been reported. Recently, we experienced a case of R. radiobacter bacteremia in a patient who underwent chemotherapy for lymphoma. Here, we report the case with a review of the literature.
Agrobacterium tumefaciens ; Bacteremia ; Endocarditis ; Humans ; Korea ; Lymphoma ; Rhizobium ; Soil

Actin filaments play an important role in fungal life processes such as growth, development and cytokinesis. The expression vector pSULPH-Lifeact-mCherry of fluorescent mCherry-labeled actin was transferred into Verticillium dahliae Kleb. wild type V592 by the genetic transformation system mediated by Agrobacterium tumefaciens to obtain the stable fluorescent labeled actin strain V592/Lifeact-mCherry. Then we detected its biological phenotype and the dynamic changes of actin fluorescence during the process of spore germination, mycelial growth and development. There was no significant difference in the colony morphology, colonial growth rate, sporulation and germination rate between the fluorescent labeled actin strain and the wild type. The actin fluorescence signal was observed at the tip of the conidia and hyphae and the septum clearly. Actin participated in the formation of the contractile actomyosin ring (CAR) during cytokinesis by observing the dynamic behavior of the actin in the process of hyphal septum formation. The fluorescent labeled actin strain can be used to study the dynamics of actin in fungal development to provide theoretical and practical support for further study of the mechanism of actin in fungal development and pathogenesis.
Actins ; Agrobacterium tumefaciens ; Plant Diseases ; Spores, Fungal ; Verticillium

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.
Agaricales* ; Agrobacterium ; Cell Membrane ; Cell Wall ; DNA ; Protoplasts

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called T1 plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow T1 plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of T1 plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high Ca²⁺ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high Ca²⁺ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observation suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.
Agrobacterium tumefaciens* ; Agrobacterium* ; Endocytosis ; Genome, Plant ; In Vitro Techniques ; Plant Cells ; Plant Tumors ; Plants ; Plasmids ; Polyethylene Glycols ; Protoplasts* ; Spheroplasts* ; Tobacco* ; Virulence ; Wounds and Injuries

The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown gall tumors by infecting the wounded dicotyledonous plants and subsequent integration of bacterial DNA into plant nuclear DNA. Virulent A. tumefaciens strains harbor a large Ti (tumor–inducing) plasmid that carries genes essential for tumorigenesis. In the present study, 13 strains (Malus pumila Mill; A₁₋₃, Populus monilifera; W₁₋₆, Populus tomentiglandlosa; P₁₋₃ and Rosa species; R₁) of Agrobacterium isolated in korean crown gall tumors and plasmids were observed in 6 strains (W₂, W₃, W₆, P₁, P₃ and A₂). The test for crown gall tumor formation was resulted only in ATCC15955 and KW2 strains inoculated into the stem of sun flower and the development was observed for 4 and 6 weeks after inoculation. Above two Ti plasmids (pTi) were purified by cesium chloride-ethidium bromide density gradient centrifugation and digested with restriction enzyme and fragments of pTiATCC15955 and pTiKW₂ observed by EcoR I ; 25&27, Hind III ; 23&21, BamH I ; each 20 and Hpa I ; 12&27. And sizes of pTiATCC15955 and and pTiKW₂ calculated as 200 and 87 kbases. Octopine was isolated from tumor tissue (W₁₋₆ and P₁₋₃) and these strains confirmed as octopine type.
Agrobacterium tumefaciens ; Agrobacterium* ; Carcinogenesis ; Centrifugation, Density Gradient ; Cesium ; DNA ; DNA, Bacterial ; Flowers ; Korea* ; Plant Tumor-Inducing Plasmids ; Plant Tumors ; Plants ; Plasmids* ; Populus ; Rosa ; Soil ; Solar System ; Wounds and Injuries

PURPOSE: We report a case of Agrobacterium radiobacter endophthalmitis following cataract surgery and treated with pars plana vitrectomy and intravitreal antibiotics injection. METHODS: A 63 year-old male patient was transferred for the endophthalmitis following cataract surgery on his left eye. His initial visual acuity was hand motion. Slit lamp examination showed marked inflammatory cells in anterior chamber and vitreous opacity was seen in fundus examination with indirect ophthalmoscopy. We performed the removal of implanted IOL, pars plana vitrectomy and intravitreal antibiotics injection(Vancomycin 1.0 mg/0.1 ml, Ceftazidime 2.0 mg/0.1 ml). RESULTS: Gram stain results revealed the gram negative rods and bacterial culture identification revealed the Agrobacterium radiobacter using the RapID NF plus system. After 8 weeks, his corrected visual acuity is 0.3 and fundus examination shows the localized atrophic change and preretinal membrane in inferotemporal retina.
Agrobacterium tumefaciens* ; Agrobacterium* ; Anterior Chamber ; Anti-Bacterial Agents ; Cataract* ; Ceftazidime ; Endophthalmitis* ; Hand ; Humans ; Male ; Membranes ; Middle Aged ; Ophthalmoscopy ; Retina ; Visual Acuity ; Vitrectomy

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of 50~100 hygromycin B-resistant transformants per 1x10(7) conidia of A. niger. This efficiency is improved 10~20 fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 microg/ml of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.
Agar ; Agrobacterium tumefaciens* ; Agrobacterium* ; Aspergillus nidulans ; Aspergillus niger* ; Aspergillus* ; Fungi* ; Genome, Fungal ; Hygromycin B ; Niger ; Polymerase Chain Reaction ; Protoplasts ; Saccharomyces cerevisiae ; Spores, Fungal ; Yeasts