Yeast protein extraction

For protein extraction, 10 ml of co-transformed yeast strains were grown to an OD₆₀₀ of 1 in SD-UHW/Gal/Raf liquid medium at 30°C with shaking at 200 rpm. The proteins were precipitated using the ammonium acetate method (modified from Karginov and Agaphonov et al., 2016 [66]). In short, cells were harvested by centrifugation at 700 xg for 5 min. The pellets were resuspended in 200 μl NH₄-acetate buffer (1 M NH₄(CH₃COO), 150 mM NaCl, 30 mM Tris-HCl, pH 7.5, 10 mM PMSF, 5 mM EDTA, and one tablet of Protease Inhibitor Cocktail (Roche). The yeast suspension was transferred into BeadBug-prefilled tubes with 0.5-mm silica glass beads (Sigma) and ground in a Precellys homogenizer (two times at 6,200 rpm for 30 sec, break: 15 sec). Afterwards, the DNA was sheared using a Diogenode Bioruptur ultrasonic water bath (twice for 30 sec at high power, break: 90 sec). The suspension without the beads was transferred into a Protein LoBind tube (Eppendorf). The glass beads were washed three times with 250 μl NH₄-acetate buffer. The washes were combined with the suspension and incubated for 1.5 h on ice. Precipitated proteins were harvested by centrifugation (16,000 x g for 10 min). Precipitates were washed with 1 ml 1 M NaCl and the pellet was resuspended with 200 μl Urea-SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 8 M Urea, 1% ß-mercaptoethanol, 2 mM EDTA, 5% glycerol, and 0.004% bromophenol blue) at room temperature. Resuspension in urea-SDS buffer and omission of the boiling step is essential for detection of LexA-MLA fusion proteins. For western blotting, 10–15 μl of the sample were loaded on 8% or 12% SDS page gels, blotted onto PVDF membranes and probed with either anti-HA (Merck, clone 3F10, RRID:AB_390914) or anti-LexA (Santa Cruz, Biotechnology, sc7544, RRID:AB_627883) primary antibodies, followed by incubation with secondary anti-rat (Santa Cruz Biotechnology, sc2065, RRID:AB_631756) or anti-mouse IgG-HRP antibodies (Santa Cruz Biotechnology, sc2005, RRID:AB_631736) for the detection of AVR_A or MLA proteins, respectively. HA and LexA fusion proteins were detected by HRP activity on SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher 34095) using a Gel Doc XR and gel documentation system (Bio-Rad).