Orange g dye

To allow analysis under the CLSM, each sealer was mixed with Orange G dye (16230 Sigma-Aldrich, Dorset, UK) used in 0.1% concentration (n = 3). After tooth preparation, the samples were placed in a 35 mm petri dish, covered with water and examined with an inverted Leica TCS-SP8 confocal system equipped with an DM6 upright microscope and a 40x/0.80 HCX water immersion lens (Leica Microsystems GmbH, Mannheim, Baden-Württemberg, Germany) with an excitation/emission wavelength of 494/521 nm. Four randomly selected images were made per sample. The photomicrographs acquired were assessed independently by three previously trained, calibrated and blinded evaluators.

Detached leaves treated with crude enzyme solution and B. cinerea spores were incubated for 3 dpi under the same conditions. Each leaf was fixed with FAA solution (5% formaldehyde, 70% ethanol, and 5% acetic acid) for 48 h. The fixed samples were dehydrated with ethanol (70% for 24 h and 85% for 3 h) and subsequently displaced with butanol through a butanol solution series (35% for 1 h, 55% for 1 h, 75% for 1 h, and 100% for 24 h). Samples were then embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO). Tissue sections (15 μm thick) were prepared using a rotary microtome (Leitz, Austria). The samples were dewaxed in xylene, and treated sequentially with anhydrous ethanol and 70% ethanol. Then, the samples were stained according to Stoughton⁴⁴ with some modifications. First, staining was done with 0.025% (w/v) thionin acetate (Sigma-Aldrich) in 0.1 mM sodium acetate and a saturated solution of Orange G dye (Sigma-Aldrich), before rinsing with a solution of ethanol and xylene (1:1). Stained sections were mounted on glass slides and observed under a BX50 light microscope with an FX380 CCD camera system (Olympus, Tokyo, Japan).

A gel shift DNA binding assay used two complementary 50-mer oligonucleotides labeled at the 5′-end with either Cy3 or Cy5. The indicated concentration (5 or 3.6 μM) of Redβ or LiRecT in PBS (or cryo-EM buffer defined below) was mixed with 25 μM (nt) of the indicated oligonucleotide and incubated at 37 °C for 15 min. For some samples as indicated on the gel (lanes labeled “35”, “ad” or “nc”), a second oligonucleotide was added and incubated for an additional 15 min at 37 °C. For all samples the total reaction volume was 30 μl. For visualization 17.5 μl of each complex was mixed with 7.5 μl Orange G dye (65% w/v sucrose, 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.3% Orange G powder from Sigma Life Sciences), loaded onto a 0.8% agarose gel and electrophoresed in 1× TBE at room temperature for 72 min at 96 V. Gels were imaged using a Sapphire Biomolecular Imager (Azure Biosystems) with Sapphire Capture Software (version 1.12.0921.0). Scanning parameters for Fig. 8 were pixel size 100 μm, scan speed high, 2.38 mm focus, intensity 2 for Cy5, intensity 4 for Cy3, black lighting 50, white 37186, gamma 1.37. Scanning parameters for Supplementary Fig. 1a, b were intensity 1 for Cy5, intensity 2 for Cy3, black lighting 50, white 15362, gamma 0.88.