Zellutrans

Partially purified SCFE1 fractions from S. sclerotiorum strain 1946 were used^{18 (link)}. The most active fractions from eight rounds of fungal culture and the two-step cation-exchange chromatography purification protocol were pooled and dialyzed overnight in 2 l 25 mM MES, pH 7.0, at 4 °C in a dialysis membrane (ZelluTrans, nominal MWCO: 3.5; 46 mm, Roth), afterwards vacuum-filtrated through a cellulose acetate membrane (Ciro Manufacturing Corporation, pore size: 0.2μm) and loaded with a flow rate of 1 ml/min onto a Source 15S 4.6/100PE column (GE Healthcare) equilibrated with buffer A (50 mM MES, pH 5.4). After washing with buffer A, bound proteins were eluted with a linear gradient of buffer B (500 mM KCl, 50 mM MES, pH 5.4; 0–50% in 15 column volumes) and 500 μl fractions were collected using automated fractionation.

E. coli bacteria harboring the respective plasmids were incubated in LB-broth with ampicillin until an OD₆₀₀ of 0.5. Once the OD was reached, isopropyl-β-D-thiogalactopyranoside (IPTG) (VWR, catalogue #AC121) at a final concentration of 1mM was added and the cultures were then incubated at room temperature (RT) for 2.5h under constant shaking. Following centrifugation, E. coli were subjected to a lysozyme digest (1 mg/ml lysozyme 4°C 30 min) followed by ultrasound treatment (Brandson sonifier, 5mm tip, 35% amplitude, 1min 45sec total with 10 sec pulse on, 20sec pulse off). Recombinant proteins were then purified under native conditions via Ni-TED columns as recommended by the manufacturer (Macherey-Nagel, Protino Ni-TED, catalogue #745120.25) and dialyzed against phosphate buffered saline using dialysis membranes with a molecular weight cut off of 6000–8000Da (Carl Roth, ZelluTrans,catalogue #E660.1) and frozen in liquid nitrogen. Recombinant muramidase-released protein (rMrp) was purified as previously described [44]. His-tagged proteins were subjected to SDS-polyacrylamide gel electrophoresis and stained by Coomassie (expedeon, InstantBlue, catalogue #SKU: ISB1L).

Atg18^WT and mutant DNA were amplified from the corresponding pRS316 plasmids and cloned into a pEXP5‐NT/TOPO vector (Invitrogen). Plasmids were transformed into E. coli BL21. A 50 ml preculture overnight was used to inoculate 2 l of LB media (37°C). Cells were grown to an OD₆₀₀ of 0.8–0.9. Cultures were then cooled to 16°C on ice, and IPTG (Roche) was added to a final concentration of 0.2 mM. Cells were shaken overnight (200 rpm, 16°C), pelleted, washed once in ice‐cold lysis buffer (500 mM NaCl, 50 mM Tris pH 7.4, 10 mM KPi), and resuspended in one pellet volume of lysis buffer with complete EDTA‐free protease inhibitor cocktail (Roche) before purification. Purification was performed as previously described (Gopaldass et al, 2017 (link)). To conjugate the Dylight550 fluorophore (Thermo Fisher), proteins were incubated at room temperature with an equimolar amount of the dye in PBS containing 300 mM of NaCl at room temperature protected from light. To remove the excess of fluorophore proteins were dialyzed in PBS with 300 mM NaCl overnight at 4°C with a 12 kDa cutoff membrane (ZelluTrans, ROTH).

LPS was extracted from lyophilized bacteria by hot phenol–water method described by Westphal et al. [26 ]. The water phase was intensively dialyzed against deionized water for 3 days (ZelluTrans, 30 kDa MWCO; Carl Roth GmbH + Co., Karlsruhe, Germany) and lyophilized. The crude LPS was resuspended in ultrapure water, homogenized by sonication, and separated by threefold ultracentrifugation, each for 6 h at 105,000× g using Beckman Coulter centrifuge (Beckman Coulter Life Sciences Division, Indianapolis, IN, USA). The supernatant after first ultracentrifugation was collected and lyophilized, where LPS pallet was further purified according to need.