Calcofluor white m2r

Cytological analyses were conducted on 5–10 nodules originating from three different plants for each condition using the protocol described by Bonaldi et al. (2011) (link). Confocal microscopy observations were carried out using a confocal laser-scanning microscope (Carl Zeiss LSM 700; Jena, Germany). Sections of ORS278 WT and BRADO4680 nodules were incubated for 20 min in live/dead staining solution [5 μM SYTO 9 and 30 μM propidium iodide (PI) in 50 mM Tris pH 7.0 buffer; Live/Dead BacLight, Invitrogen]. All the nodule sections were incubated for 15 min in 10 mM phosphate saline buffer (PBS) containing calcofluor white M2R (Sigma, Munich) at a final concentration of 0.01% (w/v) to stain the plant cell wall (Nagata and Takebe, 1970 (link)). Calcofluor was excited at 405 nm and emission signals were collected from 405 to 470 nm. For SYTO 9 or GFP and PI, an excitation wavelength of 488 and 555 nm was used with emission signal collected at 490–522 nm and 555–700 nm, respectively. Images were obtained using the ZEN 2008 software (Zeiss).

Mitochondrial and cytosolic superoxide levels were quantified in the same manner as previously reported method [25 (link)] with some modifications. A. flavus was cultured for 24 or 48 hand mycelia were harvested by filtration, washed with distilled water, and incubated with 5 μM mitoSOX or 30 μM DHE for the detection of superoxide in mitochondria and cytosol, respectively. Then the mycelia were incubated with 3 μM Calcofluor White M2R (Sigma-Aldrich) and applied to microscopic slides. A BX53 fluorescence microscope equipped with a DP70 camera (Olympus, Tokyo, Japan) was used to capture fluorescent images. Superoxide level in a region of interest was estimated as follows: Using Image J software, the blue component of each fluorescent image of Calcofluor White M2R was binarized and the dimensions of the binarized area were regarded as mycelial mass in the image. Similarly, the red component of each fluorescent image of mitoSOX and DHE was first subjected to background subtraction and then binarized at the threshold set at “20”. The dimensions of the binarized area were regarded as superoxide amount in the image. The relative superoxide level was calculated with the equation: superoxide level in the image = superoxide amount/mycelial mass × 100. Supplementary Figure S2 shows schematic representation of quantification of superoxide in a region of interest.

For histochemical analyses, 20-μm-thick cryomicrotome sections were submerged in individual histochemical reagents. For lignin staining, 2 % phloroglucinol in 25 % HCl solution was used. For suberin staining, sections were immersed in 0.1 % (w/v) solution of Sudan Red 7B in 50 % (w/v) PEG 400 in glycerol (Brundrett et al., 1991 (link)). For detection of pectins, sections were immersed in a drop of 0.05 % (w/v) Ruthenium Red. For cellulose detection, sections were immersed in a drop of 0.1 % (v/v) Calcofluor White M2R (Sigma) and subsequently one drop of 10 % KOH was added. All reagents were applied for 1–5 min; the staining solutions were then removed and the sections were rinsed thoroughly in distilled water and observed under bright-field microscopy (staining for lignin, suberin, pectins) or confocal laser scanning microscopy (autofluorescence, staining for cellulose). The autofluorescence of the tissues was observed in sections without any staining.

CR-containing agar (CR-agar) was prepared by supplementing LB agar w/o salt with CR and Coomassie brilliant blue G-250. When indicated, CR-agar was supplemented with Ebba680 (2 μl/ml). A volume of 20 ml CR-agar was used in Petri dishes, while 2 ml was used per well in sterile, Costar tissue culture treated 6-well plates (Sigma-Aldrich, Stockholm, Sweden). Biofilm formation was initiated by positioning 10 μl aliquots from liquid, stationary phase cultures onto CR-agar in the Petri dish and in wells of the 6-well plate. After incubation at 28 °C for 3–4 days, morphotypes based on curli and cellulose expression were determined by visual inspection and documented by photography. To perform the Calcofluor assay, LB agar w/o salt supplemented with 2 μl Calcofluor White stain (Calcofluor White M2R (1 g/L), Evans blue (0.5 g/L), Sigma-Aldrich, Stockholm, Sweden) per ml agar was used in the 6-well plate format. For fluorescence-based identification of live and dead bacteria in the biofilms, LB agar w/o salt was supplemented with the Live/Dead stain using 1 μl each of SYTO®9 and propidium iodide (PI) per ml agar in the 6-well plate format. Biofilms in the Calcofluor assay and Live/Dead assay were allowed to form according to the same procedure as used in the CR assay and analysed by automated microscopy (see below).

Zymography was performed using standard SDS-PAGE gels^{58 (link)} except for containing 0.1% ethylene glycol chitin (Wako Pure Chemical Industries). We used the sample buffer without SDS and reducing agent in the sample buffer and loaded the samples onto the gel without heat denaturation. After electrophoresis, the gel was incubated in 50 mM Gly-HCl (pH 2.0) containing 1% (V/V) Triton-X-100 at 37 °C for 1 hour. After that, gels were submerged in 50 mM Tris-HCl (pH 7.6) containing 1% (V/V) Triton X-100 at 37 °C overnight. After refolding, the gel was stained with freshly prepared 0.01% (W/V) Calcofluor white M2R (Sigma-Aldrich) in 50 mM Tris-HCl (pH 7.6). After 30 min-gentle shaking, the brightener solution was alkalized for emission. The gels were analyzed using the Luminescent Image Analyzer. Then, the gel was stained with CBB.