For infection assays, X. euvesicatoria strains were resuspended in 1 mM MgCl2 at an optical density (OD600nm) of 0.1 which corresponds to 1×108 colony-forming units (CFU) ml−1. Bacterial suspensions were infiltrated into leaves of the pepper cultivar Early Cal Wonder (ECW) using a needleless syringe. Infected plants were incubated in growth chambers for 16 hours of light at 28°C and 8 hours of darkness at 22°C. Disease symptoms were photographed seven days post inoculation (dpi). In planta growth curves were performed in three ECW plants as described (Bonas et al., 1991 (link)). To monitor translocation of dTALE-2, X. euvesicatoria strains were resuspended in 1 mM MgCl2 at a density of 4×108 CFU ml−1 and infiltrated into leaves of gfp-transgenic Nicotiana benthamiana plants (Werner et al., 2011 (link)). Infected N. benthamiana plants were incubated for 16 hours of light at 20°C and 8 hours of darkness at 18°C. GFP fluorescence was documented four dpi using a chemiluminescence/fluorescence imager (Vilber Fusion FX Edge). Infection experiments were performed at least three times with different transconjugants; representative results are shown.
Fusion fx edge
Manufactured by Vilber
The Fusion FX Edge is a gel documentation system designed for imaging and analyzing nucleic acid and protein gels. It features a high-resolution camera and a range of illumination options, including UV, white, and blue light, to capture clear and accurate images of samples. The system is equipped with intuitive software for image analysis and quantification.
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Lab products found in correlation
4 protocols using fusion fx edge
1
Pepper Bacterial Infection Assays
2
In vitro T3S Assays with X. euvesicatoria
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In vitroT3S assays with X. euvesicatoria strains were performed as described previously (Rossier et al., 1999 (link)). For this, bacteria were grown overnight in minimal medium A (MA) (pH 7.0) supplemented with sucrose (10 mM) and casamino acids (0.3%) and shifted to MA (pH 5.3; T3S permissive) containing 50 μg ml−1 bovine serum albumin (BSA) and 10 μM thiamine at an optical density at 600 nm (OD600) of 0.2 for 4 h. Cultures were incubated on a rotary shaker at 30°C, and bacterial cells and secreted proteins from 6 ml-cultures were separated by filtration. Proteins in 4 ml of the culture supernatants were precipitated with trichloroacetic acid and resuspended in 20 μl of Laemmli buffer. Total cell extracts and culture supernatants were analysed by SDS-PAGE and immunoblotting, using antibodies directed against the c-Myc epitope as well as against HrpF and HrcJ, respectively (Rossier et al., 1999 (link); Büttner et al., 2002 (link)). Horseradish peroxidase-labelled anti-mouse and anti-rabbit antibodies were used as secondary antibodies. Binding of antibodies was visualized using a chemiluminescence imager (Vilber Fusion FX Edge). Experiments were performed three times with similar results.
3
In Vitro T4S Assay for X. euvesicatoria
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For in vitro T4S assays, X. euvesicatoria strains were grown over night in NYG medium with antibiotics, resuspended in fresh NYG medium containing 50 μg ml−1 bovine serum albumin (BSA) at a cell density of 2 × 108 CFU/ml, and incubated on a rotary shaker at 30°C for 4 h. Bacterial cells and secreted proteins were separated by filtration as described previously (Rossier et al., 1999 (link)). Proteins in 2 ml of the culture supernatants were precipitated with trichloroacetic acid and resuspended in 20 μl of Laemmli buffer. Total cell extracts and culture supernatants were analyzed by SDS-PAGE and immunoblotting, using antibodies directed against the c-Myc epitope (Sigma Aldrich) and the β-subunit of the RNA polymerase (Invitrogen), respectively. Horseradish peroxidase-labelled anti-mouse and anti-rabbit antibodies were used as secondary antibodies. Binding of antibodies was visualized using a chemiluminescence imager (Vilber Fusion FX Edge). Results were reproduced at least two times.
4
GST Pull-Down Assay for Protein Interactions
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For GST pull-down assays, E. coli BL21 (DE3) cells expressing the potential interaction partners under control of the lac promoter were grown in LB medium until an OD600 of 0.6–0.8 and gene expression was induced in the presence of IPTG (isopropyl-β-D-thiogalactopyranoside; 2 mM final concentration) for 2 h at 37°C. Bacterial cells were harvested by centrifugation, broken with a French press and cell debris were removed by an additional centrifugation step. GST and GST fusion proteins present in the cell lysates were immobilized on a glutathione sepharose matrix according to the manufacturer’s instructions (GE Healthcare). After washing of the matrix, immobilized GST and GST fusion proteins were incubated with bacterial lysates containing the predicted interaction partner for 2 h at 4°C on an overhead shaker. Unbound proteins were removed by washing the sepharose matrix and bound proteins were eluted with Laemmli buffer. Cell lysates and eluted proteins were analysed by SDS-PAGE and immunoblotting using antibodies directed against the c-Myc epitope and GST, respectively. Horseradish peroxidase-labelled anti-mouse and anti-goat antibodies were used as secondary antibodies. Binding of antibodies was visualized using a chemiluminescence imager (Vilber Fusion FX Edge). Experiments were performed three times with similar results.
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