Verticillium strains (Table
Agrobacterium tumefaciens‐mediated Verticillium transformation was performed as described previously (Santhanam,
Cefotaxime
Manufactured by Duchefa Biochemie
Sourced in Netherlands
Cefotaxime is a third-generation cephalosporin antibiotic. It is a broad-spectrum antibiotic effective against a wide range of Gram-positive and Gram-negative bacteria.
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Lab products found in correlation
Hygromycin, by Duchefa Biochemie (5 mentions)
Carbenicillin, by Duchefa Biochemie (2 mentions)
Kanamycin, by Duchefa Biochemie (2 mentions)
Ceftazidime, by Merck Group (2 mentions)
Maldi tof ms, by Bruker (2 mentions)
Timentin, by Duchefa Biochemie (1 mentions)
Potato dextrose broth (pdb), by Merck Group (1 mentions)
Potato dextrose broth agar, by Merck Group (1 mentions)
Phytoagar, by Duchefa Biochemie (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Mlr 351, by Sanyo (1 mentions)
Phytagel, by Merck Group (1 mentions)
Ticarcillin, by Duchefa Biochemie (1 mentions)
Gamborg b5 vitamins, by Duchefa Biochemie (1 mentions)
Tryptone, by Duchefa Biochemie (1 mentions)
Yeast extract, by Duchefa Biochemie (1 mentions)
Lr reaction kit, by Thermo Fisher Scientific (1 mentions)
Bp reaction kit, by Thermo Fisher Scientific (1 mentions)
Kinetin, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
18 protocols using cefotaxime
1
Generation of Verticillium Mutant Strains
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Verticillium strains (TableS1 ) were grown on potato dextrose agar (PDA; Oxoid, Basingstoke, UK) at 22°C. The Ave1 knockout construct pRF‐HU2_Ave1 that was described previously (de Jonge et al., 2012 ) was used to generate Ave1 deletion mutants in V. nonalfalfae strain Vna 5431 and V. dahliae strain V4 (Table S1 ). The Ave1 complementation construct pFBT 005_pAve1::Ave1 that was described earlier (Song et al., 2017b ) was used to generate Ave1 expression strains in V. alfalfae strain Va2 (Table S1 ).
Agrobacterium tumefaciens‐mediated Verticillium transformation was performed as described previously (Santhanam,2012 ), and Verticillium deletion transformants were selected on PDA (Oxoid, Basingstoke, UK) containing 200 μg/mL cefotaxime and 50 μg/mL hygromycin (Duchefa, Haarlem, the Netherlands). Ave1 expression transformants were selected on PDA supplemented with 200 μg/mL cefotaxime (Duchefa, Haarlem, the Netherlands) and 50 μg/mL nourseothricin sulphate (Sigma‐Aldrich Chemie BV, Zwijndrecht, the Netherlands). Putative Verticillium transformants were tested by PCR, and subsequent inoculation on Ve1 tomato plants (Ve1) and tomato cultivar Moneymaker plants (ve1) (Fradin et al., 2009 ), or N. glutionsa plants carrying a functional Ve1 homolog (Song et al., 2017a ).
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Verticillium strains (Table
Agrobacterium tumefaciens‐mediated Verticillium transformation was performed as described previously (Santhanam,
2
Agrobacterium-mediated Poplar Transformation
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Agrobacterium-mediated genetic transformations of poplar plants were performed by applying a leaf-disc transformation protocol [58 (link)] or with an advanced leaf disc method as described in Bruegmann et al. [59 ]. In summary, for the latter, plant tissue was infected with Agrobacterium and washed with Cefotaxime (500 mg/L, Duchefa Biochemie) and cultivated on WPM medium as described in Fladung et al. [58 (link)] and Bruegmann et al. [59 ], with thidiazuron added to a final concentration of 0.0022 mg/L ([60 (link)], Duchefa Biochemie). For selection, the regeneration medium was supplemented with Cefotaxime (500 mg/L, Duchefa Biochemie) and kanamycin (50 mg/L, Duchefa Biochemie) or hygromycin (20 mg/L, Duchefa Biochemie), depending on the selection marker gene. Until beginning stem regeneration, the batches were cultivated in the dark. Once regenerating shoots began to appear, the regenerants were transferred for one month to low light condition with 2.5 µmol photons m−2 s−1 until initial multiple stem formation and later on under standard conditions as described above. Selected regenerants were used for molecular analyses. After verification of transgenic status, the plants were propagated on WPM medium without supplements [38 ].
3
Agrobacterium-Mediated Transformation of A. evenia
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140 explants (110 epicotyls and 30 hypocotyls) were immersed for 1 h in the diluted A. tumefaciens suspension. After blot-drying between sterile filter papers, explants were placed on solidified MS medium for 3 days. The A. evenia explants were then rinsed three times, for 1 h each time, with sterile MS medium containing 250 mg/L cefotaxime (Duchefa Biochemie) to eliminate A. tumefaciens, blotted dry on sterile filter paper and placed on the MS regeneration medium containing 0.5 μM NAA and 4.4 μM BAP, 10 mg/L hygromycin and 250 mg/L cefotaxime. After the first 2 weeks of cultivation, the concentration of hygromycin was increased to 20 mg/L. Two separate controls were included in the experiment. They consisted of 40 untransformed explants (20 epicotyls and 20 hypocotyls) which were placed on MS regeneration medium either with antibiotics (control for antibiotic selection) or without antibiotic (control for regeneration). Explants were transplanted in a freshly prepared medium every 2 weeks and the apparition of contaminations was regularly checked.
4
Agrobacterium-Mediated Transformation of B. rotunda Cell Cultures
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B. rotunda cell suspension cultures were used as the target explant in this project.
Transformed Agrobacterium carrying recombinant construct was cultured overnight for 16 to 18 hours in LB broth medium containing 30mg/mL of Rifampicin and 50mg/L of Kanamycin. Approximately, 2mL of settled cell volume (SCV) of cell suspension cultures were briefly air-dried by sucking out the liquid media and left in laminar flow cabinet for 10 to 15 min. Cells were infected by bacteria suspensions at room temperature for 10 min without shaking. During the infection, 100μM of Acetosyringone (AS) was added to the MS liquid medium. After the infection, the cells were blotted dry on filter paper in a petri dish containing solid MS medium supplemented with 1mg/L of 2,4-D, 0.5mg/L of BAP and 100μM of AS. The cells were then co-cultivated for 3 days in dark condition. Next, the infected cells were cultured in liquid MS medium supplemented with 1mg/L of 2,4-D, 0.5mg/L of BAP and 300mg/L of Cefotaxime (Duchefa Biochemie, Netherlands) on a rotary shaker (100rpm) at 25±1°C for 1 week. The cells were then grown on semi-solid MS medium supplemented with 1.0mg/L of 2,4-D , 0.5mg/L of BAP, 300mg/L of Cefotaxime and 15mg/L of Hygromycin (Duchefa Biochemie, Netherlands) for selection of resistant callus in 4-week intervals, respectively.
Transformed Agrobacterium carrying recombinant construct was cultured overnight for 16 to 18 hours in LB broth medium containing 30mg/mL of Rifampicin and 50mg/L of Kanamycin. Approximately, 2mL of settled cell volume (SCV) of cell suspension cultures were briefly air-dried by sucking out the liquid media and left in laminar flow cabinet for 10 to 15 min. Cells were infected by bacteria suspensions at room temperature for 10 min without shaking. During the infection, 100μM of Acetosyringone (AS) was added to the MS liquid medium. After the infection, the cells were blotted dry on filter paper in a petri dish containing solid MS medium supplemented with 1mg/L of 2,4-D, 0.5mg/L of BAP and 100μM of AS. The cells were then co-cultivated for 3 days in dark condition. Next, the infected cells were cultured in liquid MS medium supplemented with 1mg/L of 2,4-D, 0.5mg/L of BAP and 300mg/L of Cefotaxime (Duchefa Biochemie, Netherlands) on a rotary shaker (100rpm) at 25±1°C for 1 week. The cells were then grown on semi-solid MS medium supplemented with 1.0mg/L of 2,4-D , 0.5mg/L of BAP, 300mg/L of Cefotaxime and 15mg/L of Hygromycin (Duchefa Biochemie, Netherlands) for selection of resistant callus in 4-week intervals, respectively.
5
Tobacco Transformation using Agrobacterium
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A. tumefaciens harboring pCTN containing nptII and hpt genes was used to infect N. tabacum cell suspension cultures, according to Shumin et al. [32 (link)]. The 4-day-old N. tabacum cell suspension culture (5 mL) was mixed with the A. tumefaciens culture (100 μL) and co-cultivated at 28 °C in darkness for 3 days without shaking. After incubation, the cells were washed twice with 15 mL TLM supplemented with 300 mg/L cefotaxime (Duchefa, Haarlem, The Netherlands) and 100 mg/L carbenicillin (Duchefa, Haarlem, The Netherlands). The N. tabacum cells were then resuspended and plated on selection agar TSM (TLM supplemented with 300 mg/L cefotaxime, 100 mg/L carbenicillin, 30 mg/L hygromycin B, 2% (w/v) Gelrite®). After 5–6 weeks of culture, the generated calli were transferred to TSM for propagation. A single colony of callus was selected to be propagated on liquid TSM to obtain a single cell line. The transformation efficiency was calculated and expressed as the number of positive regenerants per total number of independent transformation events.
6
In vitro propagation and transformation of potato and tomato
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The tetraploid potato cultivar Desiree (ZPC, Joure, The Netherlands) was in vitro propagated in a controlled environmental chamber at 19 °C under a 16 h light/8 h dark photoperiod and transformed as previously described [25 (link)]. Plant regeneration was performed using a selection medium containing 250 µg mL−1 cefotaxime (Duchefa, Haarlem, The Netherlands), 100 µg mL−1 timentin® (Duchefa, Haarlem, The Netherlands) and 50 µg mL−1 kanamycin (Duchefa, Haarlem, The Netherlands).
Plants from the WVA106 tomato cultivar were cultured in sterile conditions in a growth chamber with controlled temperatures of 22 °C/18 °C under a 16 h/8 h (day/night) photoperiod. Agrobacterium-mediated transformation using the C58 pGV2260 strain was performed on cotyledon segments from 8-12 day-old seedlings, as previously described [29 (link)]. Plant regeneration was performed using a selection medium containing 225 µg mL−1 timentin® (Duchefa, Haarlem, The Netherlands) and 100 µg mL−1 kanamycin (Duchefa, Haarlem, The Netherlands).
Plants from the WVA106 tomato cultivar were cultured in sterile conditions in a growth chamber with controlled temperatures of 22 °C/18 °C under a 16 h/8 h (day/night) photoperiod. Agrobacterium-mediated transformation using the C58 pGV2260 strain was performed on cotyledon segments from 8-12 day-old seedlings, as previously described [29 (link)]. Plant regeneration was performed using a selection medium containing 225 µg mL−1 timentin® (Duchefa, Haarlem, The Netherlands) and 100 µg mL−1 kanamycin (Duchefa, Haarlem, The Netherlands).
7
Isolation and Culture of Fungal Mycelium
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21–30 dpi (days post inoculation), 1–1.5 cm of primary inflorescence stem segments were cut at the base (Additional file 1 : Figure S5b), surface sterilized (5 min, 70% ethanol; 5 min 0.02% sodium hydrogen chloride solution supplemented with 0.02% Triton X-100) and thoroughly washed with germ-free water (according to [13 (link)]). These stem segments were placed on solidified Potato Dextrose Broth agar (20 g/l PDB (Sigma-Aldrich); 10 g/l phyto agar (Duchefa); Milli-Q water; autoclaved at 121 °C; supplemented after cooling to 60 °C with 500 mg/l cefotaxime (Duchefa). After incubation in darkness for 3–5 days at RT, fungal mycelium became visible.
8
Detecting Antibiotic-Resistant Bacteria
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From each participant’s sample, the rectal swab was removed and 100 μl of Cary-Blair medium were spread onto MacConkey agar plates, supplemented with cefotaxime (1 mg/L; Duchefa Biochemie, Haarlem, the Netherlands), ceftazidime (2 mg/L; Sigma Aldrich, St. Louis, US), ciprofloxacin (0,125 and 0,25 mg/L; Fluka Chemicals, Buchs, Switzerland), and one control plate without supplementation. In addition, 200 μl and 400 μl of Cary-Blair medium were added into two separate tubes with MacConkey broth supplemented with 1 mg/L cefotaxime. Agar plates and broths were incubated overnight at 35 °C. The following day, the broths were spread to MacConkey agar plates with cefotaxime (1 mg/L), and incubated overnight at 35 °C. Single colonies of E.coli or Klebsiella spp. were selected from the different plates. If multiple morphologies were observed, all unique morphotypes were selected. Species identification was performed using MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany). Samples that yielded no, or sparse growth on the MacConkey control plate, were excluded from the study.
9
Agrobacterium-mediated Transformation of Marchantia
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Experiments were performed using the M. polymorpha ecotype BoGa, which was obtained from the Botanical Garden of Osnabrück, Germany. Agrobacterium tumefaciens mediated transformation of sporelings with two constructs was carried out as previously described by Ishizaki et al. [10 (link)] with minor modifications. Differing from Ishizaki et al. [10 (link)], the separation of sporelings from A. tumefaciens after three days of co-cultivation was conducted by a 15 min sedimentation followed by decantation of the added washing solution (½ Gamborg B5 medium with vitamins, Duchefa). Sedimentation and decantation were repeated four times and the sporelings were then plated on agar plates with ½ Gamborg B5 medium with vitamins supplemented with 10 µg/ml hygromycin (Sigma-Aldrich), 5 µg/ml G418 (Sigma-Aldrich) and 100 µg/ml cefotaxime (Duchefa). Gemmae were investigated from the G1 up to the G4 generation. Plants were grown under sterile conditions on solid medium (1% agar medium and ½ Gamborg B5 medium with vitamins, Duchefa) in 10 × 10 × 2 cm petri dishes (Sarstedt, Nümbrecht, Germany) under a 16 h/8 h day/night regimen at 22 °C in climate cabinets (Sanyo MLR351).
10
Hairy Root Transformation Protocol
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Seeds were sterilized by chlorine gas for six hours, followed by one hour of ventilation in sterile conditions, cold stratified at 4 °C for 24 h, and germinated in plant tissue culture boxes containing 50% Murashige and Skoog (MS) medium (Duchefa) with 5 g/L sucrose (Penta) for the indicated time (4 or 6 weeks for A. thaliana, 4 weeks for C. hirsuta) at 21 °C, 16-h light/8-h dark photoperiod and 150 μmol/m2/s light intensity. Hairy roots were obtained by transforming plants with the transconjugant Ti-less A. tumefaciens C58C1 carrying a hairy-root-inducing plasmid pRiA4b [43 (link)]. The agrobacterial suspension was grown for 18 h in Luria-Broth medium (10 g/L of tryptone [Duchefa], 5 g/L of yeast extract [Duchefa], 5 g/L of sodium chloride [Lachner], pH = 7.0) at 28 °C and injected with an insulin syringe into the basal part of the inflorescence stem. After 2–4 cultivation weeks, hairy roots emerging from the inoculation sites were excised and placed on a solid MS medium, including Gamborg B5 vitamins (MS + B5, Duchefa; 4.4 g/L), 0.3% phytagel (Sigma) and 30 g/L sucrose (Penta), supplemented with ticarcillin (500 mg/L, Duchefa) and cefotaxime (200 mg/L, Duchefa) to eliminate agrobacteria growth. Hairy roots were grown on Petri dishes at 24 °C in the dark and transferred to fresh MS + B5 media after 5–6 weeks of cultivation.
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