Image scanner

The WinRHIZO technique was adopted to assess the root phenotype data of soybean seedlings as reported earlier [101 (link)]. Soybean roots from various experimental groups were acquired and fully spread on the image scanner (Epson, Tokyo, Japan). Test materials were assessed with the assistance of the software of WinRHIZO (WinRHIZO 2013e Professional Edition, WinRhizo Pro, Quebec, QC, Canada).

The biofilm assay was performed according to (Saising et al., 2012 (link)) with modifications. Overnight cultures of staphylococcal strains grown in TSB with an additional 0.25% glucose were adjusted to OD₅₇₈ of 0.1. 20 μl of the bacterial suspension was added to 180 μl of TSB in wells of a 96 well flat bottom microtiter plate (Greiner Bio-One), resulting in a final OD₅₇₈ of 0.01 which corresponds to approximately 10⁶ CFU/ml. The microtiter plate was incubated at 37°C without agitation for 24 h. After 24 h, the culture supernatant was discarded and the wells were rinsed twice with 200 μl of PBS before being air dried for 30 min. Then, the wells were stain with 200 μl of 01% crystal violet for 30 min and subsequently rinsed with purified water (MilliQ). The plate was air dried again for 30 min and image of the wells was taken with an image scanner (Epson). Three independent biological replicates were performed for this assay.

The MW distributions of PPI and PPIH were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [41 (link)]. SDS-PAGE analysis of PPI and PPIH was performed with a 12.5% separation gel and 5% stacking gel. PPI (6 mg) and PPIH (6 mg) were separately mixed with 1 mL of buffer (0.02% bromophenol blue, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 70 mM Tris-HCl, pH 6.8). Next, the samples were heated at 95 °C for 7 min. Samples (10 μL) and protein ladders (6 μL) were loaded into separate wells. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. After staining, the gel was washed with 10% acetic acid to destain. The stained gel was digitized using an image scanner (Epson America Inc., Long Beach, CA, USA). PPIH was also analyzed by an Autoflex III mass spectrometer (Bruker Daltonik, Bremen, Germany). The masses in the range of 0–500 were measured. Each sample was analyzed in triplicate.